Methods and compositions for treatment of myotubular myopathy using chimeric polypeptides comprising myotubularin 1(MTM1) polypeptides

ABSTRACT

The present invention provides chimeric polypeptides comprising myotubularin 1 (MTMI) polypeptides and an internalising moiety, wherein, the moiety can be an antibody, and is preferably monoclonal antibody 3E10, a functional variant or a fragment thereof. One aspect of the present invention provides compositions comprising these chimeric polypeptides together with a pharmaceutically acceptable carrier, and optionally, a further therapeutic agent. Another aspect of the present invention provides methods of treating Myotubular Myopathy comprising administering the polypeptides or compositions comprising the polypeptides to a subject in need.

RELATED APPLICATIONS

This application is a national stage filing under 35 U.S.C. 371 ofInternational Application No. PCT/US2010/038703, filed Jun. 15, 2010,which claims the benefit of the filing date under 35 U.S.C. 119(e) toUnited States provisional application number 61/268,732, filed June 15,2009, the entire content of which is hereby incorporated by reference.International application Ser. No. PCT/US2010/038703 was published underPCT Article 21(2) in English.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted via EFS-Web and is hereby incorporated by reference in itsentirety. Said ASCII copy, created on May 2, 2012, is named1061990003301Seq.TXT, and is 38,792 bytes in size.

BACKGROUND OF THE INVENTION

Myotubular myopathy (MTM) is a rare and severe X-linked muscle disorderthat occurs with an estimated incidence of 1 male in every 50,000births. Myotubular myopathy is a member of a category of diseasesreferred to as centronuclear myopathies. A cardinal feature ofcentronuclear myopathies is that the nucleus is positioned in the centerof many of the affected individual's muscle cells, rather than in thenormal location at the ends of these cells. Although centronuclearmyopathies share this characteristic feature, the various diseases havedifferent causes, afflict different patient populations, and have uniquedisease progression and prognosis.

Myotubular myopathy is caused by a deficiency of the myotubularin 1(MTM1) protein, a phosphoinositide phosphatase (Bello A B et al., HumanMolecular Genetics, 2008, Vol. 17, No. 14). At birth MTM patientspresent with severe hypotonia and respiratory distress and those thatsurvive the neonatal period are often totally or partially dependentupon ventilator support (Taylor G S et al., Proc Natl Acad Sci USA. 2000August 1; 97(16):8910-5; Bello A B et al., Proc Natl Acad Sci USA. 2002Nov. 12; 99(23):15060-5; Pierson C R et al., Neuromuscul Disord. 2007July; 17(7): 562-568; Herman G E et al., THE JOURNAL OF PEDIATRICSVOLUME 134, NUMBER 2). Patients with MTM exhibit delayed motormilestones and are susceptible to complications such as scoliosis,malocclusion, pyloric stenosis, spherocytosis, and gall and kidneystones, yet linear growth and intelligence are normal and the diseasefollows a non-progressive course (Herman G E et al., THE JOURNAL OFPEDIATRICS VOLUME 134, NUMBER 2). The average hospital stay for neonatalMTM patients is ˜90 days. However, patients that survive will requirelong-term ventilatory assistance and in-home care. The cost of basicsupportive care, as well as the costs associated with handling themedical complications that often arise in MTM patients, impose asubstantial personal and economic burden on patients and families.

SUMMARY OF THE INVENTION

Currently, there are no therapies for MTM. Treatment is limited toventilatory assistance and other forms of supportive care to attempt tomanage the disabilities associated with the disease. The presentdisclosure provides methods and compositions for treating MTM.

The present disclosure provides chimeric polypeptides comprising amyotubularin (MTM1) polypeptide or a bioactive fragment thereof and aninternalizing moiety, as well as compositions comprising the chimericpolypeptides in combination with a pharmaceutical carrier. Alsodisclosed are constructs useful for producing such chimericpolypeptides. Further, the present disclosure teaches methods of makingthe chimeric polypeptides and constructs that encode them. Additionally,disclosed herein are methods of using the chimeric polypeptides, forexample, to manipulate phosphatase activity in a cell and as part of atreatment of diseases or conditions associated with MTM1 mutation ordeficiency.

In one aspect, the present disclosure provides a chimeric polypeptidecomprising (i) a myotubularin (MTM1) polypeptide, or a bioactivefragment thereof and (ii) an internalizing moiety. In certainembodiments, the chimeric polypeptide has phosphoinositide phosphataseactivity. That is, the chimeric polypeptide has the ability to cleave orhydrolyze a phosphorylated phosphoinositide molecule. In certainembodiments, a substrate for the chimeric polypeptide is PI3 or PIP3. Incertain embodiments the internalizing moiety promotes transport of saidchimeric polypeptide into muscle cells. In other words, theinternalizing moiety helps the chimeric polypeptide effectively andefficiently transit cellular membranes. In some embodiments, theinternalizing moiety transits cellular membranes via an ENT2transporter. In other words, the internalizing moiety promotes transportof the chimeric polypeptide across cellular membranes via an ENT2transporter.

In certain embodiments, the MTM1 polypeptide disclosed herein comprisesan amino acid sequence at least 90% identical to SEQ ID NO: 1, or abioactive fragment thereof. In some embodiments, the MTM1 polypeptidecomprises an amino acid sequence at least 80%, 85%, 90%, 95%, 97%, 98%,99% or 100% identical to an MTM1 polypeptide (such as the MTM1polypeptides represented in one or more of SEQ ID NOs: 1, 6, 8, or abioactive fragment of any of the foregoing). In certain embodiments, theMTM1 polypeptide comprises an amino acid sequence at least 80%, 85%,90%, 95%, 97%, 98%, 99% or 100% identical to an MTM1 polypeptiderepresented in SEQ ID NOs: 1. In certain embodiments, any of theforegoing or following MTM1 polypeptides disclosed herein and for use ina chimeric polypeptide further comprise one or more polypeptide portionsthat enhance one or more of in vivo stability, in vivo half life,uptake/administration, and/or purification. In certain embodiments, anyof the foregoing or following MTM1 polypeptides and/or chimericpolypeptides may further include one or more epitope tags. Such epitopetags may be joined to the MTM1 polypeptide and/or the internalizingmoiety. When more than one epitope tag is present (e.g., 2, 3, 4) thetags may be the same or different.

In some embodiments, the internalizing moiety of any of the foregoingchimeric polypeptides comprises an antibody or an antigen-bindingfragment thereof. In certain embodiments, the antibody is a monoclonalantibody or an antigen-binding fragment thereof. The antibody orantigen-binding fragment thereof may be, e.g., monoclonal antibody 3E10,or a variant thereof that retains the cell penetrating activity of 3E10,or an antigen-binding fragment of 3E10 or said 3E10 variant. In certainembodiments, the antibody or antigen-binding fragment thereof may be anantibody or antigen-binding fragment that binds to the same epitope as3E10, or an antibody or antigen-binding fragment that has substantiallythe same cell penetrating activity as 3E10, or an antigen-bindingfragment thereof. In other embodiments, the internalizing moiety of anyof the foregoing chimeric polypeptides comprises a homing peptide asdescribed herein. In certain embodiments, the internalizing moietycomprises an antibody or antigen-binding fragment comprising a lightchain variable domain (VL) comprising an amino acid sequence at least90%, 92%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 4. Incertain embodiments, the internalizing moiety comprises an antibody orantigen-binding fragment comprising a heavy chain variable domain (VH)comprising an amino acid sequence at least 90%, 92%, 95%, 96%, 97%, 98%,99%, or 100% identical to SEQ ID NO: 2. In certain embodiments, theinternalizing moiety comprises an antibody or antigen-binding fragmentcomprising: a light chain variable domain (VL) comprising an amino acidsequence at least 90%, 92%, 95%, 96%, 97%, 98%, 99%, or 100% identicalto SEQ ID NO: 4 and a heavy chain variable domain (VH) comprising anamino acid sequence at least 90%, 92%, 95%, 96%, 97%, 98%, 99%, or 100%identical to SEQ ID NO: 2. The invention specifically contemplatesinternalizing moieties based on any combination of the foregoing VH andVL chains, for example, an internalizing moiety comprising a VHcomprising an amino acid sequence at least 98% identical to SEQ ID NO: 2and a VL at least 96% identical to SEQ ID NO: 4. In certain embodiments,the internalizing moiety comprising a VH comprising the amino acidsequence set forth in SEQ ID NO: 2 and a VL comprising the amino acidsequence set forth in SEQ ID NO: 4. As detailed herein, the VH and VLdomains may be included as part of a full length antibody or as part ofa fragment, such as an scFv. Moreover, the VH and VL domains may bejoined by a linker, or may be joined directly. In either case, the VHand VL domains may be joined in either orientation (e.g., with the VLdomain N-terminal to the VH domain or with the VH domain N-terminal tothe VL domain).

In certain embodiments, any of the foregoing chimeric polypeptides maybe produced by chemically conjugating the MTM1 polypeptide, or bioactivefragment thereof, to the internalizing moiety. In certain embodiments,the chimeric polypeptide may be produced recombinantly to recombinantlyconjugate the MTM1 polypeptide, or bioactive fragment thereof, to theinternalizing moiety. For example, the chimeric polypeptide may beproduced using a recombinant vector encoding both the MTM1 polypeptideand the internalizing moiety. In some embodiments, the chimericpolypeptide is produced in a prokaryotic or eukaryotic cell. Forexample, the eukaryotic cell may be selected from a yeast cell, an aviancell, an insect cell, or a mammalian cell. Note that for embodiments inwhich the MTM1 polypeptide is chemically conjugated to the internalizingmoiety, the invention contemplates that the MTM1 polypeptide and/orinternalizing moiety may be produced recombinantly.

In some embodiments, the MTM1 polypeptide or bioactive fragment thereofmay be conjugated or joined (whether chemically or recombinantly) to theinternalizing moiety by a linker. In other embodiments, the MTM1polypeptide or bioactive fragment thereof may be conjugated or joineddirectly to the internalizing moiety. For example, a recombinantlyconjugated chimeric polypeptide can be produced as an in-frame fusion ofthe MTM1 portion and the internalizing moiety portion. In certainembodiments, the linker may be a cleavable linker. In any of theforegoing embodiments, the internalizing moiety may be conjugated(directly or via a linker) to the N-terminal or C-terminal amino acid ofthe MTM1 polypeptide. In other embodiments, the internalizing moiety maybe conjugated (directly or indirectly) to an internal amino acid of theMTM1 polypeptide. Note that the two portions of the construct areconjugated/joined to each other. Unless otherwise specified, describingthe chimeric polypeptide as a conjugation of the MTM1 portion to theinternalizing moiety is used equivalently as a conjugation of theinternalizing moiety to the MTM1 portion. In certain embodiments, alinker joins together one or more portions of the internalizing moiety,such as a VH and VL domain of an antibody. The invention contemplatesthe use of 0 linkers, 1 linker, 2 linkers, and more than two linkers.When more than 1 linker is used, the linkers may be the same ordifferent.

In certain embodiments, any of the foregoing chimeric polypeptides maybe formulated as compositions formulated in a pharmaceuticallyacceptable carrier. In certain embodiments, the compositions areformulated for intravenous administration.

In a related aspect, the disclose provides chimeric polypeptidescomprising the amino acid sequence set forth in SEQ ID NO: 11 or theamino acid sequence set forth in SEQ ID NO: 11, but in the absence ofone or both epitope tags. Such chimeric polypeptides, as well as any ofthe chimeric polypeptides described herein, may be used in any of themethods described herein.

With respect to chimeric polypeptides, the disclosure contemplates allcombinations of any of the foregoing aspects and embodiments, as well ascombinations with any of the embodiments set forth in the detaileddescription and examples. Further, any of the chimeric polypeptides ofthe disclosure can be used in any of the methods described herein.

In another aspect, the present disclosure provides a nucleic acidconstruct, comprising a nucleotide sequence that encodes an MTM1polypeptide or a bioactive fragment thereof, operably linked to anucleotide sequence that encodes an internalizing moiety. In certainembodiments, the nucleic acid construct encodes a chimeric polypeptidehaving phosphoinositide phosphatase activity. In certain embodiments,the internalizing moiety targets muscle cells to promote transport intomuscle cells. In other embodiments, the internalizing moiety transitscellular membranes via an ENT2 transporter.

In some embodiments, the nucleotide sequence that encodes an MTM1polypeptide comprises a nucleotide sequence at least 90% identical toSEQ ID NO: 5. In certain embodiments, the nucleotide sequence thatencodes an MTM1 polypeptide comprises a nucleotide sequence at least80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to any one or moreof SEQ ID NOs: 5, 7, or 9. In certain embodiments, the nucleotidesequence is a nucleotide sequence that encodes an MTM1 polypeptidecomprising an amino acid sequence at least 80%, 85%, 90%, 95%, 97%, 98%,99% or 100% identical to any one or more of SEQ ID NOs: 1, 6, or 8. Incertain embodiments, the nucleotide sequence is a nucleotide sequencethat encodes an MTM1 polypeptide comprising an amino acid sequence atleast 90%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 1.

In certain embodiments, the nucleic acid constructs may further comprisea nucleotide sequence that encodes a linker.

In certain embodiments, the internalizing moiety may be an antibody oran antigen-binding fragment thereof. In some embodiments, the antibodyor antigen-binding fragment thereof may be monoclonal antibody 3E10, ora variant thereof that retains the cell penetrating activity of 3E10, oran antigen-binding fragment of 3E10 or said 3E10 variant. In otherembodiments, the antibody or antigen-binding fragment thereof may be anantibody or antigen-binding fragment that binds to the same epitope as3E10, or an antibody or antigen-binding fragment that has substantiallythe same cell penetrating activity as 3E10, or an antigen-bindingfragment thereof. In certain embodiments, the internalizing moiety maybe a homing peptide. In certain embodiments, the internalizing moiety isa homing peptide which targets muscle cells.

In another aspect, the present disclosure provides a compositioncomprising any of the foregoing chimeric polypeptide compositions ornucleic acid constructs, and a pharmaceutically acceptable carrier. Incertain embodiments, the composition may further comprise a second agentwhich acts in an additive or synergistic manner for treating myotubularmyopathy, for having a bioactive effect of MTM1 on cells, and/or forpromoting transport into cells. The second agent may be, e.g., a smallmolecule, a polypeptide, an antibody, an antisense oligonucleotide, oran siRNA molecule.

With respect to nucleic acid constructs, the disclosure contemplates allcombinations of any of the foregoing aspects and embodiments, as well ascombinations with any of the embodiments set forth in the detaileddescription and examples.

In further aspects, the present disclosure provides a method of treatingmyotubular myopathy in a subject in need thereof, comprisingadministering to the subject an effective amount of any of the foregoingchimeric polypeptides or nucleic acid constructs. In certainembodiments, the method comprising administering a chimeric polypeptide,which polypeptides comprise: (i) an MTM1 polypeptide or bioactivefragment thereof and (ii) an internalizing moiety which promotestransport of said chimeric polypeptide into muscle cells. In certainembodiments, the chimeric polypeptide has phosphoinositide phosphataseactivity. In certain embodiments, the subject is a human. In otherembodiments, the subject is selected from any of a mouse, rat, ornon-human primate.

In some embodiments, the internalizing moiety transits cellularmembranes via an ENT2 transporter. In other words, the internalizingmoiety promotes transport into cells via an ENT2 transporter. In certainembodiments, the internalizing moiety comprises an antibody or anantigen-binding fragment thereof. In certain embodiments, theinternalizing moiety comprises a monoclonal antibody or anantigen-binding fragment thereof. For example, the antibody orantigen-binding fragment thereof may be monoclonal antibody 3E10, or avariant thereof that retains the cell penetrating activity of 3E10, oran antigen-binding fragment of 3E10 or said 3E10 variant. Additionally,the antibody or antigen-binding fragment thereof may be an antibody orantigen-binding fragment that binds to the same epitope as 3E10, or anantibody or antigen-binding fragment that has substantially the samecell penetrating activity as 3E10, or an antigen-binding fragmentthereof.

In certain embodiments, the MTM1 polypeptide comprises an amino acidsequence at least 90% identical to SEQ ID NO: 1, or a bioactive fragmentthereof. In some embodiments the MTM1 polypeptide comprises an aminoacid sequence at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100%identical to an MTM1 polypeptide (such as the MTM1 polypeptidesrepresented in one or more of SEQ ID NOs: 1, 6, 8, or a bioactivefragment of any of the foregoing).

In some embodiments, any of the foregoing chimeric polypeptides may beformulated with a pharmaceutically acceptable carrier.

In certain embodiments, the chimeric polypeptides for use in the claimedmethod may be conjugated (e.g., chemically or recombinantly) asdescribed herein.

In certain embodiments, any of the foregoing methods may furthercomprise a second therapy which acts in an additive or synergisticmanner for treating myotubular myopathy. In some embodiments, the secondtherapy may be a drug for helping to relieve one or more symptoms ofmyotubular myopathy, or a physical or other non-drug therapy fortreating or otherwise helping to relieve one or more symptoms ofmyotubular myopathy. Exemplary non-drug therapies include, but are notlimited to, ventilatory therapy, occupational therapy, acupuncture, andmassage.

In some embodiments, any of the foregoing chimeric polypeptides may beadministered via an appropriate route of administration, e.g.,systemically, locally, or intravenously. In certain embodiments, thechimeric polypeptide is administered intravenously via bolus injectionor infusion.

With respect to methods for treating myotubular myopathy, the disclosurecontemplates all combinations of any of the foregoing aspects andembodiments, as well as combinations with any of the embodiments setforth in the detailed description and examples.

In another aspect, the present disclosure provides a method ofdelivering a chimeric polypeptide or nucleic acid construct into a cellvia an equilibrative nucleoside transporter (ENT2) pathway, comprisingcontacting a cell with a chimeric polypeptide or nucleic acid construct.In certain embodiments, the method comprises contacting a cell with achimeric polypeptide, which chimeric polypeptide comprises an MTM1polypeptide or bioactive fragment thereof and an internalizing moietywhich mediates transport across a cellular membrane via an ENT2 pathway,thereby delivering the chimeric polypeptide into the cell. In certainembodiments, the cell is a muscle cell.

In certain embodiments, the MTM1 polypeptide comprises an amino acidsequence at least 90% identical to SEQ ID NO: 1, or a bioactive fragmentthereof. In some embodiments the MTM1 polypeptide comprises an aminoacid sequence at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100%identical to an MTM1 polypeptide (such as the MTM1 polypeptidesrepresented in one or more of SEQ ID NOs: 1, 6, 8, or a bioactivefragment of any of the foregoing).

In certain embodiments, the MTM1 polypeptide may further comprise one ormore polypeptide portions that enhance one or more of in vivo stability,in vivo half life, uptake/administration, and/or purification. In otherembodiments, the internalizing moiety comprises an antibody or anantigen-binding fragment thereof. In other embodiments, theinternalizing moiety comprises a monoclonal antibody or anantigen-binding fragment thereof. For example, the antibody orantigen-binding fragment thereof may be monoclonal antibody 3E10, or avariant thereof that retains the cell penetrating activity of 3E10, oran antigen-binding fragment of 3E10 or said 3E10 variant. Additionally,the antibody or antigen-binding fragment thereof may be an antibody orantigen-binding fragment that binds to the same epitope as 3E10, or anantibody or antigen-binding fragment that has substantially the samecell penetrating activity as 3E10, or an antigen-binding fragmentthereof. In some embodiments, the internalizing moiety may comprise ahoming peptide that targets ENT2.

In certain embodiments, the chimeric polypeptides for use in the methodmay be produced by chemically conjugating the MTM1 polypeptide, orbioactive fragment thereof, to the internalizing moiety. In someembodiments, the chimeric polypeptide may be produced recombinantly torecombinantly conjugate the MTM1 polypeptide, or bioactive fragmentthereof, to the internalizing moiety. In certain embodiments, thechimeric polypeptides for use in the claimed method may be conjugated(e.g., chemically or recombinantly) as described herein.

With respect to methods of delivering a chimeric polypeptide into a cellvia an equilibrative nucleoside transporter (ENT2) pathway, thedisclosure contemplates all combinations of any of the foregoing aspectsand embodiments, as well as combinations with any of the embodiments setforth in the detailed description and examples.

In another aspect, the present disclosure provides a method ofdelivering a chimeric polypeptide into a muscle cell, comprisingcontacting a muscle cell with a chimeric polypeptide or nucleic acidconstruct. In certain embodiments, the method comprises contacting themuscle cell with a chimeric polypeptide, which chimeric polypeptidecomprises an MTM1 polypeptide or a bioactive fragment thereof and aninternalizing moiety which promotes transport into muscle cells, therebydelivering the chimeric polypeptide into the muscle cell. In certainembodiments, the internalizing moiety promotes transport via anequilibrative nucleoside transporter (ENT2) pathway

In certain embodiments, the MTM1 polypeptide comprises an amino acidsequence at least 90% identical to SEQ ID NO: 1, or a bioactive fragmentthereof. In some embodiments the MTM1 polypeptide comprises an aminoacid sequence at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100%identical to an MTM1 polypeptide (such as the MTM1 polypeptidesrepresented in one or more of SEQ ID NOs: 1, 6, 8, or a bioactivefragment thereof).

In certain embodiments, the MTM1 polypeptide may further comprise one ormore polypeptide portions that enhance one or more of in vivo stability,in vivo half life, uptake/administration, and/or purification. In otherembodiments, the internalizing moiety comprises an antibody or anantigen-binding fragment thereof. In other embodiments, theinternalizing moiety comprises a monoclonal antibody or anantigen-binding fragment thereof. For example, the antibody orantigen-binding fragment thereof may be monoclonal antibody 3E10, or avariant thereof that retains the cell penetrating activity of 3E10, oran antigen-binding fragment of 3E10 or said 3E10 variant. Additionally,the antibody or antigen-binding fragment thereof may be an antibody orantigen-binding fragment that binds to the same epitope as 3E10, or anantibody or antigen-binding fragment that has substantially the samecell penetrating activity as 3E10, or an antigen-binding fragmentthereof. In some embodiments, the internalizing moiety may comprise ahoming peptide that targets ENT2, and/or a homing peptide that targetsmuscle cells.

In some embodiments, the chimeric polypeptide of any of the foregoingmethods may be produced by chemically conjugating the MTM1 polypeptide,or bioactive fragment thereof, to the internalizing moiety. In otherembodiments, the chimeric polypeptide may be produced recombinantly torecombinantly conjugate the MTM1 polypeptide, or bioactive fragmentthereof, to the internalizing moiety. In certain embodiments, thechimeric polypeptides for use in the claimed method may be conjugated(e.g., chemically or recombinantly) as described herein.

With respect to methods of delivering a chimeric polypeptide into a cellvia an equilibrative nucleoside transporter (ENT2) pathway, thedisclosure contemplates all combinations of any of the foregoing aspectsand embodiments, as well as combinations with any of the embodiments setforth in the detailed description and examples.

In other aspects, the present disclosure provides a method of deliveringa polypeptide to a subject in need thereof, comprising administering toa subject in need thereof a chimeric polypeptide or a nucleic acidconstruct. In certain embodiments, the method comprises administering achimeric polypeptide, which chimeric polypeptide comprises an MTM1polypeptide or a bioactive fragment thereof and an internalizing moietywhich promotes transport into muscle cells, thereby delivering thechimeric polypeptide into the muscle cell. In certain embodiments, theinternalizing moiety promotes transport via an ENT2 transporter.

In certain embodiments, the MTM1 polypeptide comprises an amino acidsequence at least 90% identical to SEQ ID NO: 1, or a bioactive fragmentthereof. In some embodiments the MTM1 polypeptide comprises an aminoacid sequence at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100%identical to an MTM1 polypeptide (such as the MTM1 polypeptidesrepresented in one or more of SEQ ID NOs: 1, 6, 8, or a bioactivefragment thereof).

In certain embodiments, the MTM1 polypeptide may further comprise one ormore polypeptide portions that enhance one or more of in vivo stability,in vivo half life, uptake/administration, and/or purification. In otherembodiments, the internalizing moiety comprises an antibody or anantigen-binding fragment thereof. In other embodiments, theinternalizing moiety comprises a monoclonal antibody or anantigen-binding fragment thereof. For example, the antibody orantigen-binding fragment thereof may be monoclonal antibody 3E10, or avariant thereof that retains the cell penetrating activity of 3E10, oran antigen-binding fragment of 3E10 or said 3E10 variant. Additionally,the antibody or antigen-binding fragment thereof may be an antibody orantigen-binding fragment that binds to the same epitope as 3E10, or anantibody or antigen-binding fragment that has substantially the samecell penetrating activity as 3E10, or an antigen-binding fragmentthereof. In some embodiments, the internalizing moiety may comprise ahoming peptide that targets ENT2, and/or targets muscle cells.

In some embodiments, the chimeric polypeptide of any of the foregoingmethods may be produced by chemically conjugating the MTM1 polypeptide,or bioactive fragment thereof, to the internalizing moiety. In otherembodiments, the chimeric polypeptide may be produced recombinantly torecombinantly conjugate the MTM1 polypeptide, or bioactive fragmentthereof, to the internalizing moiety. In certain embodiments, thechimeric polypeptides for use in the claimed method may be conjugated(e.g., chemically or recombinantly) as described herein.

In certain embodiments, the subject of any of the foregoing methods maybe a human. In some embodiments, the method of delivery may be, e.g.,parenteral or intravenous. In certain embodiments, the chimericpolypeptide is administered intravenously, for example, via bolusinjection or infusion.

With respect to methods of delivering a chimeric polypeptide into musclecells, the disclosure contemplates all combinations of any of theforegoing aspects and embodiments, as well as combinations with any ofthe embodiments set forth in the detailed description and examples.

In another aspect, the present disclosure provides a method ofincreasing MTM1 bioactivity in a muscle cell, comprising contacting amuscle cell with a chimeric polypeptide, which chimeric polypeptidecomprises an MTM1 polypeptide or bioactive fragment thereof and aninternalizing moiety which promotes transport into muscle cells, therebyincreasing MTM1 bioactivity in the muscle cell. In certain embodiments,the internalizing moiety promotes transport via an ENT2 transporter.

In certain embodiments, the MTM1 polypeptide comprises an amino acidsequence at least 90% identical to SEQ ID NO: 1, or a bioactive fragmentthereof. In some embodiments the MTM1 polypeptide comprises an aminoacid sequence at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100%identical to an MTM1 polypeptide (such as the MTM1 polypeptidesrepresented in one or more of SEQ ID NOs: 1, 6, 8, or bioactivefragments of any of the foregoing).

In certain embodiments, the MTM1 polypeptide may further comprise one ormore polypeptide portions that enhance one or more of in vivo stability,in vivo half life, uptake/administration, and/or purification. In otherembodiments, the internalizing moiety comprises an antibody or anantigen-binding fragment thereof. In other embodiments, theinternalizing moiety comprises a monoclonal antibody or anantigen-binding fragment thereof. For example, the antibody orantigen-binding fragment thereof may be monoclonal antibody 3E10, or avariant thereof that retains the cell penetrating activity of 3E10, oran antigen-binding fragment of 3E10 or said 3E10 variant. Additionally,the antibody or antigen-binding fragment thereof may be an antibody orantigen-binding fragment that binds to the same epitope as 3E10, or anantibody or antigen-binding fragment that has substantially the samecell penetrating activity as 3E10, or an antigen-binding fragmentthereof. In some embodiments, the internalizing moiety may comprise ahoming peptide that targets ENT2, and/or muscle cells.

In other embodiments, the chimeric polypeptide for use in any of theforegoing methods may be produced by chemically conjugating the MTM1polypeptide, or bioactive fragment thereof, to the internalizing moiety.In other embodiments, the chimeric polypeptide may be producedrecombinantly to recombinantly conjugate the MTM1 polypeptide, orbioactive fragment thereof, to the internalizing moiety.

In some embodiments, the MTM1 bioactivity includes, e.g., MTM1phosphoinositide phosphatase activity, or MTM1 association with anendosomal protein, or both. In certain embodiments, the phosphoinositideactivity is at least 50% that of native MTM1, or at least 80% that ofnative MTM1. In other embodiments, the phosphoinositide activity is atleast 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% that ofnative MTM1. Bioactivity can be assessed relative to that in a control.

With respect to methods of increasing MTM1 bioactivity in cells, thedisclosure contemplates all combinations of any of the foregoing aspectsand embodiments, as well as combinations with any of the embodiments setforth in the detailed description and examples. The foregoing methodsbased on administering chimeric polypeptides or contacting cells withchimeric polypeptides can be performed in vitro (e.g., in cells orculture) or in vivo (e.g., in a patient or animal model). In certainembodiments, the method is an in vitro method. In certain embodiments,the method is an in vivo method.

In other aspects, the present disclosure also provides a method ofproducing any of the foregoing chimeric polypeptides as describedherein. Further, the present disclosure contemplates any number ofcombinations of the foregoing methods and compositions.

The invention contemplates all combinations of any of the foregoingaspects and embodiments, as well as combinations with any of theembodiments set forth in the detailed description and examples.

BRIEF DESCRIPTION OF THE TABLES

Table 1. An experimental design to evaluate phosphoinositide phosphataseactivity, endosomal association and secretion of genetically conjugatedFv3E10-G53-hMTM1. Certain predicted results for control groups areindicated by “yes” or “no”; “?” indicates results to-be-measured. “+” or“−” indicates the presence or absence of a particular compound to thesample. “*” indicates that the prediction is based upon the assumptionthat phosphoinositide activities will be a function of endogenous andhMTM1 dependent activity.

Table 2. An experimental design to evaluate if Fv3E10-G53-hMTM1 enterscells via ENTs and associates with endosomal proteins. See Table 1description for notations. “*” indicates that the sample isimmunoprecipitated and detected by immunoblot only if immunoprecipitatedin Table 1, groups 10 through 18. “**” indicates that prediction isbased on the assumption that the genetic conjugate has no defects inassociation between Vps34 and hMTM1.

Table 3. In vivo dosing plan for chemically and genetically conjugated3E10-MTM1. Dosing is planned for twice per week over 20 weeks. Blood andtissues will be collected for immunohistochemistry (IHC), hematoxylinand eosin staining (H&E), and protein isolation.

DETAILED DESCRIPTION OF THE INVENTION

Proteins of the MTM family fall into two basic categories: familymembers that exhibit phosphoinositide phosphatase activity and familymembers that bind phosphoinositides but are catalytically inactive.MTM1, mutations in which result in myotubular myopathy, is catalyticallyactive and possesses phosphoinositide phosphatase activity. Someexamples of phosphoinositides that act as substrates for MTM1 include,but are not limited to, e.g., phosphatidylinositol 3-phosphate (PI(3)P),PI(4)P, PI(5)P, PI(3,4)P₂, PI(4,5)P₂, PI(3,5)P₂, PI(3,4,5)P₃, as well assynthetic phosphoinositide compounds useful for in vitro assays. Incertain embodiments, the chimeric polypeptide is capable of cleaving orhydrolyzing any one or more of the foregoing phosphoinositides. Incertain embodiments, the chimeric polypeptide is capable of cleaving orhydrolyzing PIP3.

MTM1, as well as other related MTM proteins (MTMRs) assembleindividually or in heterodimers on endocytic vesicles at various stagesof subcellular transport. MTM1 associates with MTMR12 and interacts withother endosomal proteins such as the GTPase Rab5 and the PI 3-kinasehVps34 via the hVps15 adapter molecule. Without being bound by theory,the differential recruitment and opposing activities of MTM1 PIP3phosphatase and hVps34 PI-3 kinase may coordinate the temporal membranedistribution of PI and PIP3 that directs the intracellular trafficpatterns of endocytic vesicles. Although other MTM-related proteinspossess phosphoinositide phosphatase activity, their subcellularlocalization is sufficiently non-overlapping from that of MTM 1 thatthey are unable to functionally compensate for the MTM1 deficiency. MTM1is ubiquitously expressed yet the absence of MTM1 in skeletal musclesolely accounts for the pathophysiology of MTM (Taylor G S et al., ProcNatl Acad Sci USA. 2000 August 1; 97(16):8910-5; Bello A B et al., ProcNatl Acad Sci USA. 2002 Nov. 12; 99(23):15060-5), and suggests that thephosphoinositide phosphatase activity of MTM1 possesses a uniquesubcellular function that is particularly crucial to normal skeletalmuscle function. It is believed that MTM1 participates in themaintenance of the longitudinal and transverse architecture of theT-tubule system, and thus defects in the organization of thesestructures would impair excitation-contraction coupling, and result inthe ensuing muscle weakness and atrophy that are hallmarks of thedisease (Bello A B et al., Human Molecular Genetics, 2008, Vol. 17, No.14; Laporte J et al., HUMAN MUTATION 15:393.409 (2000); Herman G E etal., THE JOURNAL OF PEDIATRICS VOLUME 134, NUMBER 2; Weisbart R H etal., J. Immunol. 2000 June 1; 164(11):6020-6).

In certain aspects, the disclosure provides conjugates of MTM1 (e.g.,chimeric polypeptides comprising MTM1 or a bioactive fragment thereof)that may be used to treat conditions associated with MTM1 deficiency,e.g., myotubular myopathy. The terms “polypeptide,” “peptide” and“protein” are used interchangeably herein to refer to a polymer of aminoacid residues. The terms apply to amino acid polymers in which one ormore amino acid residue is an artificial chemical mimetic of acorresponding naturally occurring amino acid, as well as to naturallyoccurring amino acid polymers and non-naturally occurring amino acidpolymers.

I. MTM1 Polypeptides

As used herein, the MTM1 polypeptides include various splicing isoforms,variants, fusion proteins, and modified forms of the wildtype MTM1polypeptide. Such isoforms, bioactive fragments or variants, fusionproteins, and modified forms of the MTM1 polypeptides have at least aportion of the amino acid sequence of substantial sequence identity tothe native MTM1 protein, and retain at least one function of the nativeMTM1 protein. In certain embodiments, a bioactive fragment, variant, orfusion protein of an MTM1 polypeptide comprises an amino acid sequencethat is at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical toan MTM1 polypeptide (such as the MTM1 polypeptides represented in one ormore of SEQ ID NOs: 1, 6, and 8). As used herein, “fragments” areunderstood to include bioactive fragments or bioactive variants thatexhibit “bioactivity” as described herein. That is, bioactive fragmentsor variants of MTM1 exhibit bioactivity that can be measured and tested.For example, bioactive fragments or variants exhibit the same orsubstantially the same bioactivity as native (i.e., wild-type, ornormal) MTM1 protein, and such bioactivity can be assessed by theability of the fragment or variant to, e.g., cleave or hydrolyze anendogenous phosphoinositide substrate known in the art, or an artificialphosphoinositide substrate for in vitro assays (i.e., a phosphoinositidephosphatase activity), recruit and/or associate with other proteins suchas, for example, the GTPase Rab5, the PI 3-kinase Vps34 or Vps15 (i.e.,proper localization), or treat myotubular myopathy. Methods in which toassess any of these criteria are described herein. As used herein,“substantially the same” refers to any parameter (e.g., activity) thatis at least 70% of a control against which the parameter is measured. Incertain embodiments, “substantially the same” also refers to anyparameter (e.g., activity) that is at least 75%, 80%, 85%, 90%, 92%,95%, 97%, 98%, 99%, 100%, 102%, 105%, or 110% of a control against whichthe parameter is measured.

The structure and various motifs of the MTM1 polypeptide have been wellcharacterized in the art (see, e.g., Laporte et al., 2003, HumanMolecular Genetics, 12(2):R285-R292; Laporte et al., 2002, Journal ofCell Science 15:3105-3117; Lorenzo et al., 2006, 119:2953-2959). Assuch, in certain embodiments, various bioactive fragments or variants ofthe MTM1 polypeptides can be designed and identified by screeningpolypeptides made, for example, recombinantly from the correspondingfragment of the nucleic acid encoding an MTM1 polypeptide. For example,several domains of MTM1 have been shown to be important for itsphosphatase activity or localization. To illustrate, these domainsinclude: Glucosyltransferase, Rab-like GTPase Activator andMyotubularins (GRAM; amino acid positions 29-97 or up to 160 of SEQ IDNO: 1), Rac-Induced recruitment Domain (RID; amino acid positions161-272 of SEQ ID NO: 1), PTP/DSP homology (amino acid positions 273-471of SEQ ID NO: 1; catalytic cysteine is amino acid 375 of SEQ ID NO: 1),and SET-interacting domain (SID; amino acid positions 435-486 of SEQ IDNO: 1). Accordingly, any combination of such domains may be constructedto identify fragments or variants of MTM1 that exhibit the same orsubstantially the same bioactivity as native MTM1. Suitable bioactivefragments can be used to make chimeric polypeptides, and such chimericpolypeptides can be used in any of the methods described herein.

Exemplary fragments that may be used as part of a chimeric polypeptideinclude, for example: about residues 29-486 of SEQ ID NO: 1. Thus, incertain embodiments, the chimeric polypeptides comprises residues 29-486of SEQ ID NO: 1.

In certain embodiments, the MTM1 portion of the chimeric polypeptidecorresponds to the sequence of human MTM1. For example, the MTM1 portionof the chimeric polypeptide comprises an amino acid sequence at least90%, 92%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 1.

In addition, fragments or variants can be chemically synthesized usingtechniques known in the art such as conventional Merrifield solid phasef-Moc or t-Boc chemistry. The fragments or variants can be produced(recombinantly or by chemical synthesis) and tested to identify thosefragments or variants that can function as well as or substantiallysimilarly to a native MTM1 protein, for example, by testing theirability to cleave or hydrolyze a endogenous phosphoinositide substrateor a synthetic phosphoinositide substrate (i.e., phosphoinositidephosphatase activity), recruit and/or associate with other proteins suchas, for example, GTPase Rab5, PI 3-kinase hVps34 or hVps15 (i.e., properlocalization), or treat myotubular myopathy.

In certain embodiments, the present invention contemplates modifying thestructure of an MTM1 polypeptide for such purposes as enhancingtherapeutic or prophylactic efficacy, or stability (e.g., ex vivo shelflife and resistance to proteolytic degradation in vivo). Such modifiedMTM1 polypeptides have the same or substantially the same bioactivity asnaturally-occurring (i.e., native or wild-type) MTM1 polypeptide.Modified polypeptides can be produced, for instance, by amino acidsubstitution, deletion, or addition. For instance, it is reasonable toexpect, for example, that an isolated replacement of a leucine with anisoleucine or valine, an aspartate with a glutamate, a threonine with aserine, or a similar replacement of an amino acid with a structurallyrelated amino acid (e.g., conservative mutations) will not have a majoreffect on the biological activity of the resulting molecule.Conservative replacements are those that take place within a family ofamino acids that are related in their side chains.

This invention further contemplates generating sets of combinatorialmutants of an MTM1 polypeptide, as well as truncation mutants, and isespecially useful for identifying bioactive variant sequences.Combinatorially-derived variants can be generated which have a selectivepotency relative to a naturally occurring MTM1 polypeptide. Likewise,mutagenesis can give rise to variants which have intracellularhalf-lives dramatically different than the corresponding wild-type MTM1polypeptide. For example, the altered protein can be rendered eithermore stable or less stable to proteolytic degradation or other cellularprocess which result in destruction of, or otherwise inactivation of theprotein of interest. Such variants can be utilized to alter the MTM1polypeptide level by modulating their half-life. There are many ways bywhich the library of potential MTM1 variants sequences can be generated,for example, from a degenerate oligonucleotide sequence. Chemicalsynthesis of a degenerate gene sequence can be carried out in anautomatic DNA synthesizer, and the synthetic genes then be ligated intoan appropriate gene for expression. The purpose of a degenerate set ofgenes is to provide, in one mixture, all of the sequences encoding thedesired set of potential polypeptide sequences. The synthesis ofdegenerate oligonucleotides is well known in the art (see for example,Narang, S A (1983) Tetrahedron 39:3; Itakura et al., (1981) RecombinantDNA, Proc. 3rd Cleveland Sympos. Macromolecules, ed. A G Walton,Amsterdam: Elsevier pp 273-289; Itakura et al., (1984) Annu Rev.Biochem. 53:323; Itakura et al., (1984) Science 198:1056; Ike et al.,(1983) Nucleic Acid Res. 11:477). Such techniques have been employed inthe directed evolution of other proteins (see, for example, Scott etal., (1990) Science 249:386-390; Roberts et al., (1992) PNAS USA89:2429-2433; Devlin et al., (1990) Science 249: 404-406; Cwirla et al.,(1990) PNAS USA 87: 6378-6382; as well as U.S. Pat. Nos. 5,223,409,5,198,346, and 5,096,815).

Alternatively, other forms of mutagenesis can be utilized to generate acombinatorial library. For example, MTM1 polypeptide variants can begenerated and isolated from a library by screening using, for example,alanine scanning mutagenesis and the like (Ruf et al., (1994)Biochemistry 33:1565-1572; Wang et al., (1994) J. Biol. Chem.269:3095-3099; Balint et al., (1993) Gene 137:109-118; Grodberg et al.,(1993) Eur. J. Biochem. 218:597-601; Nagashima et al., (1993) J. Biol.Chem. 268:2888-2892; Lowman et al., (1991) Biochemistry 30:10832-10838;and Cunningham et al., (1989) Science 244:1081-1085), by linker scanningmutagenesis (Gustin et al., (1993) Virology 193:653-660; Brown et al.,(1992) Mol. Cell. Biol. 12:2644-2652; McKnight et al., (1982) Science232:316); by saturation mutagenesis (Meyers et al., (1986) Science232:613); by PCR mutagenesis (Leung et al., (1989) Method Cell Mol Biol1:11-19); or by random mutagenesis, including chemical mutagenesis, etc.(Miller et al., (1992) A Short Course in Bacterial Genetics, CSHL Press,Cold Spring Harbor, N.Y.; and Greener et al., (1994) Strategies in MolBiol 7:32-34). Linker scanning mutagenesis, particularly in acombinatorial setting, is an attractive method for identifying truncated(bioactive) forms of the MTM1 polypeptide.

A wide range of techniques are known in the art for screening geneproducts of combinatorial libraries made by point mutations andtruncations, and, for that matter, for screening cDNA libraries for geneproducts having a certain property. Such techniques will be generallyadaptable for rapid screening of the gene libraries generated by thecombinatorial mutagenesis of the MTM1 polypeptides. The most widely usedtechniques for screening large gene libraries typically comprisescloning the gene library into replicable expression vectors,transforming appropriate cells with the resulting library of vectors,and expressing the combinatorial genes under conditions in whichdetection of a desired activity facilitates relatively easy isolation ofthe vector encoding the gene whose product was detected. Each of theillustrative assays described below are amenable to high through-putanalysis as necessary to screen large numbers of degenerate sequencescreated by combinatorial mutagenesis techniques.

In certain embodiments, an MTM1 polypeptide may include a peptide and apeptidomimetic. As used herein, the term “peptidomimetic” includeschemically modified peptides and peptide-like molecules that containnon-naturally occurring amino acids, peptoids, and the like.Peptidomimetics provide various advantages over a peptide, includingenhanced stability when administered to a subject. Methods foridentifying a peptidomimetic are well known in the art and include thescreening of databases that contain libraries of potentialpeptidomimetics. For example, the Cambridge Structural Database containsa collection of greater than 300,000 compounds that have known crystalstructures (Allen et al., Acta Crystallogr. Section B, 35:2331 (1979)).Where no crystal structure of a target molecule is available, astructure can be generated using, for example, the program CONCORD(Rusinko et al., J. Chem. Inf. Comput. Sci. 29:251 (1989)). Anotherdatabase, the Available Chemicals Directory (Molecular Design Limited,Informations Systems; San Leandro Calif.), contains about 100,000compounds that are commercially available and also can be searched toidentify potential peptidomimetics of the MTM1 polypeptides.

In certain embodiments, an MTM1 polypeptide may further comprisepost-translational modifications. Exemplary post-translational proteinmodification include phosphorylation, acetylation, methylation,ADP-ribosylation, ubiquitination, glycosylation, carbonylation,sumoylation, biotinylation or addition of a polypeptide side chain or ofa hydrophobic group. As a result, the modified MTM1 polypeptides maycontain non-amino acid elements, such as lipids, poly- ormono-saccharide, and phosphates. Effects of such non-amino acid elementson the functionality of an MTM1 polypeptide may be tested for itsbiological activity, for example, its ability to treat myotubularmyopathy or ability to cleave phosphoinositides (e.g., PIP3). Given thatthe native MTM1 polypeptide is glycosylated, in certain embodiments anMTM1 polypeptide used in a chimeric polypeptide according to the presentdisclosure is glycosylated. In certain embodiments, the level andpattern of glycosylation is the same as or substantially the same asthat of the native MTM1 polypeptide. In other embodiments, the leveland/or pattern of glycosylation differs from that of the native MTM1polypeptide (e.g., underglycosylated, overglycosylated, notglycosylated).

In one specific embodiment of the present invention, an MTM1 polypeptidemay be modified with nonproteinaceous polymers. In one specificembodiment, the polymer is polyethylene glycol (“PEG”), polypropyleneglycol, or polyoxyalkylenes, in the manner as set forth in U.S. Pat.Nos. 4,640,835; 4,496,689; 4,301,144; 4,670,417; 4,791,192 or 4,179,337.PEG is a well-known, water soluble polymer that is commerciallyavailable or can be prepared by ring-opening polymerization of ethyleneglycol according to methods well known in the art (Sandler and Karo,Polymer Synthesis, Academic Press, New York, Vol. 3, pages 138-161).

In certain embodiments, fragments or variants of the MTM1 polypeptidewill preferably retain at least 50%, 60%, 70%, 80%, 85%, 90%, 95% or100% of the biological activity associated with the native MTM1polypeptide. In certain embodiments, fragments or variants of the MTM1polypeptide have a half-life (t_(1/2)) which is enhanced relative to thehalf-life of the native protein. For embodiments in which the half-lifeis enhanced, the half-life of MTM1 fragments or variants is enhanced byat least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 125%, 150%,175%, 200%, 250%, 300%, 400% or 500%, or even by 1000% relative to thehalf-life of the native MTM1 protein. In some embodiments, the proteinhalf-life is determined in vitro, such as in a buffered saline solutionor in serum. In other embodiments, the protein half-life is an in vivohalf life, such as the half-life of the protein in the serum or otherbodily fluid of an animal.

In certain aspects, an MTM1 polypeptide may be a fusion protein whichfurther comprises one or more fusion domains. Well known examples ofsuch fusion domains include, but are not limited to, polyhistidine,Glu-Glu, glutathione S transferase (GST), thioredoxin, protein A,protein G, and an immunoglobulin heavy chain constant region (Fc),maltose binding protein (MBP), which are particularly useful forisolation of the fusion proteins by affinity chromatography. For thepurpose of affinity purification, relevant matrices for affinitychromatography, such as glutathione-, amylase-, and nickel- orcobalt-conjugated resins are used. Fusion domains also include “epitopetags,” which are usually short peptide sequences for which a specificantibody is available. Well known epitope tags for which specificmonoclonal antibodies are readily available include FLAG, influenzavirus haemagglutinin (HA), and c-myc tags. In some cases, the fusiondomains have a protease cleavage site, such as for Factor Xa orThrombin, which allows the relevant protease to partially digest thefusion proteins and thereby liberate the recombinant proteins therefrom.The liberated proteins can then be isolated from the fusion domain bysubsequent chromatographic separation. In certain embodiments, the MTM1polypeptides may contain one or more modifications that are capable ofstabilizing the polypeptides. For example, such modifications enhancethe in vitro half life of the polypeptides, enhance circulatory halflife of the polypeptides or reducing proteolytic degradation of thepolypeptides. It should be noted that any portion of a chimericpolypeptide of the invention may be similarly epitope tagged. In otherwords, an epitope tag may be to MTM1 and/or the internalizing moiety.Moreover, the chimeric polypeptides may comprises more than one epitopetags, such as 2 epitope tags, or may include 0 epitope tags.

In some embodiments, an MTM1 protein may be a fusion protein with all ora portion of an Fc region of an immunoglobulin. Similarly, in certainembodiments, all or a portion of an Fc region of an immunoglobulin canbe used as a linker to link an MTM1 protein to an internalizing moiety.As is known, each immunoglobulin heavy chain constant region comprisesfour or five domains. The domains are named sequentially as follows:CH1-hinge-CH2—CH3(—CH4). The DNA sequences of the heavy chain domainshave cross-homology among the immunoglobulin classes, e.g., the CH2domain of IgG is homologous to the CH2 domain of IgA and IgD, and to theCH3 domain of IgM and IgE. As used herein, the term, “immunoglobulin Fcregion” is understood to mean the carboxyl-terminal portion of animmunoglobulin chain constant region, preferably an immunoglobulin heavychain constant region, or a portion thereof. For example, animmunoglobulin Fc region may comprise 1) a CH1 domain, a CH2 domain, anda CH3 domain, 2) a CH1 domain and a CH2 domain, 3) a CH1 domain and aCH3 domain, 4) a CH2 domain and a CH3 domain, or 5) a combination of twoor more domains and an immunoglobulin hinge region. In a preferredembodiment the immunoglobulin Fc region comprises at least animmunoglobulin hinge region a CH2 domain and a CH3 domain, andpreferably lacks the CH1 domain. In one embodiment, the class ofimmunoglobulin from which the heavy chain constant region is derived isIgG (Igγ) (γ subclasses 1, 2, 3, or 4). Other classes of immunoglobulin,IgA (Igα), IgD (Igδ), IgE (Igε) and IgM (Igμ), may be used. The choiceof appropriate immunoglobulin heavy chain constant regions is discussedin detail in U.S. Pat. Nos. 5,541,087, and 5,726,044. The choice ofparticular immunoglobulin heavy chain constant region sequences fromcertain immunoglobulin classes and subclasses to achieve a particularresult is considered to be within the level of skill in the art. Theportion of the DNA construct encoding the immunoglobulin Fc regionpreferably comprises at least a portion of a hinge domain, andpreferably at least a portion of a CH₃ domain of Fc γ or the homologousdomains in any of IgA, IgD, IgE, or IgM. Furthermore, it is contemplatedthat substitution or deletion of amino acids within the immunoglobulinheavy chain constant regions may be useful in the practice of theinvention. One example would be to introduce amino acid substitutions inthe upper CH2 region to create a Fc variant with reduced affinity for Fcreceptors (Cole et al. (1997) J. IMMUNOL. 159:3613). One of ordinaryskill in the art can prepare such constructs using well known molecularbiology techniques.

II. Internalizing Moieties

As used herein, the term “internalizing moiety” refers to a moietycapable of interacting with a target tissue or a cell type to effectdelivery of the attached molecule into the cell (i.e., penetrate desiredcell; transport across a cellular membrane). In certain embodiments,this disclosure relates to an internalizing moiety which selectively,although not necessarily exclusively, targets and penetrates musclecells. In certain embodiments, the internalizing moiety has limitedcross-reactivity, and thus preferentially targets a particular cell ortissue type. In certain embodiments, suitable internalizing moietiesinclude, for example, antibodies, monoclonal antibodies, or derivativesor analogs thereof. Other internalizing moieties include for example,homing peptides, fusion proteins, receptors, ligands, aptamers,peptidomimetics, and any member of a specific binding pair. In certainembodiments, the internalizing moiety mediates transit across cellularmembranes via an ENT2 transporter.

(a) Antibodies

In certain aspects, an internalizing moiety may comprise an antibody,such as a monoclonal antibody, a polyclonal antibody, and a humanizedantibody. Without being bound by theory, such antibody can bind to anantigen of a target tissue and thus mediate the delivery of the subjectchimeric polypeptide to the target tissue (e.g., muscle). In someembodiments, internalizing moieties may comprise antibody fragments,derivatives or analogs thereof, including without limitation: Fvfragments, single chain Fv (scFv) fragments, Fab′ fragments, F(ab′)2fragments, single domain antibodies, humanized antibodies and antibodyfragments, and multivalent versions of the foregoing. Multivalentinternalizing moieties including without limitation: monospecific orbispecific antibodies, such as disulfide stabilized Fv fragments, scFvtandems ((scFv)₂ fragments), diabodies, tribodies or tetrabodies, whichtypically are covalently linked or otherwise stabilized (i.e., leucinezipper or helix stabilized) scFv fragments; receptor molecules whichnaturally interact with a desired target molecule. In certainembodiments, the antibodies or variants thereof, may be modified to makethem less immunogenic when administered to a subject. For example, ifthe subject is human, the antibody may be “humanized”; where thecomplimentarity determining region(s) of the hybridoma-derived antibodyhas been transplanted into a human monoclonal antibody, for example asdescribed in Jones, P. et al. (1986), Nature, 321, 522-525 or Tempest etal. (1991), Biotechnology, 9, 266-273. Also, transgenic mice, or othermammals, may be used to express humanized antibodies. Such humanizationmay be partial or complete. In certain embodiments, although theantibody is a murine or other non-human antibody, its humanness score issufficient that humanization is not necessary. In still otherembodiments, the antibody or antigen-binding fragment is fully human.

In certain specific embodiments, the internalizing moiety comprises themonoclonal antibody 3E10, an antigen-binding fragment thereof, or asingle chain Fv fragment thereof. As used herein, the term “antibodies”refers to complete antibodies or antibody fragments capable of bindingto a selected target. Included are Fv, scFv, Fab′ and F(ab′)2,monoclonal and polyclonal antibodies, engineered antibodies, andsynthetic or semi-synthetic antibodies produced using phage display oralternative techniques. Monoclonal antibody 3E10 can be reproducedrecombinantly or by a hybridoma placed permanently on deposit with theAmerican Type Culture Collection (ATCC) under ATCC accession numberPTA-2439 (See U.S. Pat. No. 7,189,396). Additional suitable antibodieshave similar or substantially the same membrane penetrating activity as3E10 and/or bind to the same epitope as 3E10 and/or have substantiallythe same antigen-binding characteristics as 3E10.

Monoclonal antibody 3E10 has been shown to penetrate cells withouttoxicity and has attracted considerable interest as a means to deliverproteins and nucleic acids into the cytoplasmic or nuclear spaces oftarget tissues (Weisbart R H et al., J. Autoimmun. 1998 October;11(5):539-46; Weisbart R H, et al. Mol. Immunol. 2003 March;39(13):783-9; Zack D J et al., J. Immunol. 1996 Sep. 1; 157(5):2082-8.).Further, the VH and Vk sequences of 3E10 are highly homologous to humanantibodies, with respective humanness z-scores of 0.943 and −0.880.Thus, Fv3E10 is expected to induce less of an anti-antibody responsethan many other approved humanized antibodies (Abhinandan K R et al.,Mol. Biol. 2007 369, 852-862). A single chain Fv fragment of 3E10possesses all the cell penetrating capabilities of the originalmonoclonal antibody, and proteins such as catalase, dystrophin, HSP70and p53 retain their activity following conjugation to Fv3E10 (Hansen JE et al., Brain Res. 2006 May 9; 1088(1):187-96; Weisbart R H et al.,Cancer Lett. 2003 Jun. 10; 195(2):211-9; Weisbart R H et al., J DrugTarget. 2005 February; 13(2):81-7; Weisbart R H et al., J. Immunol. 2000June 1; 164(11):6020-6; Hansen J E et al., J Biol. Chem. 2007 July 20;282(29):20790-3). The 3E10 is built on the antibody scaffold present inall mammals; a mouse variable heavy chain and variable kappa lightchain. 3E10 gains entry to cells via the ENT2 nucleotide transporterthat is particularly enriched in skeletal muscle and cancer cells, andin vitro studies have shown that 3E10 is nontoxic. (Weisbart R H et al.,Mol. Immunol. 2003 March; 39(13):783-9; Pennycooke M et al., BiochemBiophys Res Commun. 2001 January 26; 280(3):951-9). Given the affinityof 3E10 and fragments thereof for skeletal muscle, and the ability ofvarious conjugates of 3E10 and MTM1 to maintain their respectiveactivities, a recombinant 3E10-MTM1 (and other conjugate variants asdescribed herein) therapy represents a valuable approach to treat MTM.As described herein, a recombinant 3E10 or a fragment or variant can bechemically or genetically conjugated to human MTM1 (hMTM1) and theactivity of each conjugate may be confirmed in vitro. Further, thepurified conjugates may be injected into MTM1 deficient mice andimprovements in disease phenotype, as described herein, may be examined.

The internalizing moiety may also include mutants of mAb 3E10, such asvariants of 3E10 which retain the same or substantially the same cellpenetration characteristics as mAb 3E10, as well as variants modified bymutation to improve the utility thereof (e.g., improved ability totarget specific cell types, improved ability to penetrate the cellmembrane, improved ability to localize to the cellular DNA, improvedbinding affinity, and the like). Such mutants include variants whereinone or more conservative substitutions are introduced into the heavychain, the light chain and/or the constant region(s) of the antibody.Numerous variants of mAb 3E10 have been characterized in, e.g., U.S.Pat. No. 7,189,396 and WO 2008/091911, the teachings of which areincorporated by reference herein in their entirety. In certainembodiments, the internalizing moiety comprises an antibody having anamino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 99%, or 100%identical to the amino acid sequence of 3E10, or at least 80%, 85%, 90%,95%, 96%, 97%, 99%, or 100% identical to the amino acid sequence of asingle chain Fv of 3E10 (for example, a single chain Fv comprising SEQID NO: 2 and SEQ ID NO: 4). In certain embodiments, the internalizingmoiety comprises a single chain Fv of 3E10, and the amino acid sequenceof the V_(H) domain is at least 90%, 95%, 96%, 97%, 98%, 99%, or 100%identical to SEQ ID NO: 2, and amino acid sequence of the V_(L) domainis at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ IDNO: 4. The variant 3E10 or fragment thereof retains the function of aninternalizing moiety.

In certain embodiments, the internalizing moiety comprises an antibodyor antigen-binding fragment comprising a light chain variable domain(VL) comprising an amino acid sequence at least 90%, 92%, 95%, 96%, 97%,98%, 99%, or 100% identical to SEQ ID NO: 4. In certain embodiments, theinternalizing moiety comprises an antibody or antigen-binding fragmentcomprising a heavy chain variable domain (VH) comprising an amino acidsequence at least 90%, 92%, 95%, 96%, 97%, 98%, 99%, or 100% identicalto SEQ ID NO: 2. In certain embodiments, the internalizing moietycomprises an antibody or antigen-binding fragment comprising: a lightchain variable domain (VL) comprising an amino acid sequence at least90%, 92%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 4 ANDa heavy chain variable domain (VH) comprising an amino acid sequence atleast 90%, 92%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO:2. The invention specifically contemplates internalizing moieties basedon any combination of the foregoing VH and VL chains, for example, aninternalizing moiety comprising a VH comprising an amino acid sequenceat least 98% identical to SEQ ID NO: 2 and a VL at least 96% identicalto SEQ ID NO: 4. In certain embodiments, the internalizing moietycomprising a VH comprising the amino acid sequence set forth in SEQ IDNO: 2 and a VL comprising the amino acid sequence set forth in SEQ IDNO: 4. As detailed herein, the VH and VL domains may be included as partof a full length antibody or as part of a fragment, such as an svFv.Moreover, the VH and VL domains may be joined by a linker, or may bejoined directly. In either case, the VH and VL domains may be joined ineither orientation (e.g., with the VL domain N-terminal to the VH domainor with the VH domain N-terminal to the VL domain).

As readily recognized by those of skill in the art, altered mAb 3E10(e.g., chimeric, humanized, CDR-grafted, fully human, bifunctional,antibody polypeptide dimers—i.e., an association of two polypeptidechain components of an antibody, such as one arm of an antibodycomprising a heavy chain and a light chain, or an Fab fragmentcomprising V_(L), V_(H), C_(L) and C_(H1) antibody domains, or an Fvfragment comprising a V_(L) domain and a V_(H) domain—single chainantibodies—e.g., an scFv fragment comprising a V_(L) domain linked to aV_(H) domain by a linker, and the like)—can also be produced by methodswell known in the art. Such antibodies can also be produced byhybridoma, chemical synthesis or recombinant methods described, forexample, in (Sambrook et al., Molecular Cloning: A Laboratory Manual 2dEd. (Cold Spring Harbor Laboratory, 1989); incorporated herein byreference and Harlow and Lane, Antibodies: A Laboratory Manual (ColdSpring Harbor Laboratory 1988), which is incorporated herein byreference). Moreover, other antibody internalizing moieties can bereadily made and include rodent, chimeric, humanized, fully human, etc.

Preparation of antibodies or fragments thereof (e.g., an single chain Fvfragment encoded by V_(H)-linker-V_(L)) is well known in the art. Inparticular, methods of recombinant production of mAb 3E10 antibodyfragments as well as conjugates thereof (e.g., Fv3E10-G53-hMTM1, asdisclosed herein) have been described in WO 2008/091911. Further,methods of generating scFv fragments of antibodies are well known in theart. The exemplary method of the present disclosure uses a (GGGGS)3linker (SEQ ID NO: 3) to join a 3E10 VL and VH domain. However, it isunderstood that other linkers may also be designed. For example, typicalsurface amino acids in flexible protein regions include Gly, Asn andSer. Permutations of amino acid sequences containing Gly, Asn and Serwould be expected to satisfy the criteria (e.g., flexible with minimalhydrophobic or charged character) for a linker sequence. Other nearneutral amino acids, such as Thr and Ala, can also be used in the linkersequence. In a specific embodiment, a linker sequence length of about 15amino acids can be used to provide a suitable separation of functionalprotein domains, although longer or shorter linker sequences may also beused. Moreover, it is understood that, in certain embodiments, achimeric polypeptide may include an additional linker joining theinternalizing moiety to the MTM polypeptide portion of the chimericpolypeptide. Thus, in certain embodiments, chimeric polypeptides mayinclude more than one linker, such as two linkers. For embodiments inwhich the chimeric polypeptide includes more than one linker, it isunderstood that the linkers are independently selected and may be thesame or different.

Preparation of antibodies and fragments thereof may also be accomplishedby any number of well-known methods for generating monoclonalantibodies. These methods typically include the step of immunization ofanimals, typically mice, with a desired immunogen (e.g., a desiredtarget molecule or fragment thereof). Once the mice have been immunized,and preferably boosted one or more times with the desired immunogen(s),monoclonal antibody-producing hybridomas may be prepared and screenedaccording to well known methods (see, for example, Kuby, Janis,Immunology, Third Edition, pp. 131-139, W.H. Freeman & Co. (1997), for ageneral overview of monoclonal antibody production, that portion ofwhich is incorporated herein by reference). Over the past severaldecades, antibody production has become extremely robust. In vitromethods that combine antibody recognition and phage display techniquesallow one to amplify and select antibodies with very specific bindingcapabilities. See, for example, Holt, L. J. et al., “The Use ofRecombinant Antibodies in Proteomics,” Current Opinion in Biotechnology,2000, 11:445-449, incorporated herein by reference. These methodstypically are much less cumbersome than preparation of hybridomas bytraditional monoclonal antibody preparation methods. In one embodiment,phage display technology may be used to generate an internalizing moietyspecific for a desired target molecule. An immune response to a selectedimmunogen is elicited in an animal (such as a mouse, rabbit, goat orother animal) and the response is boosted to expand theimmunogen-specific B-cell population. Messenger RNA is isolated fromthose B-cells, or optionally a monoclonal or polyclonal hybridomapopulation. The mRNA is reverse-transcribed by known methods usingeither a poly-A primer or murine immunoglobulin-specific primer(s),typically specific to sequences adjacent to the desired V_(H) and V_(L)chains, to yield cDNA. The desired V_(H) and V_(L) chains are amplifiedby polymerase chain reaction (PCR) typically using V_(H) and V_(L)specific primer sets, and are ligated together, separated by a linker.V_(H) and V_(L) specific primer sets are commercially available, forinstance from Stratagene, Inc. of La Jolla, Calif. AssembledV_(H)-linker-V_(L) product (encoding an scFv fragment) is selected forand amplified by PCR. Restriction sites are introduced into the ends ofthe V_(H)-linker-V_(L) product by PCR with primers including restrictionsites and the scFv fragment is inserted into a suitable expressionvector (typically a plasmid) for phage display. Other fragments, such asan Fab′ fragment, may be cloned into phage display vectors for surfaceexpression on phage particles. The phage may be any phage, such aslambda, but typically is a filamentous phage, such as fd and M13,typically M13. In certain embodiments, an antibody or antibody fragmentis made recombinantly. In other words, once the sequence of the antibodyis know (for example, using methods described above), the antibody canbe made recombinantly using standard techniques. Thus, other antibodiesand antigen-binding fragments related to 3E10 or with similar cellpenetrating/transiting characteristics can be readily identified byscreening, for example, a phage display library.

In certain embodiments, the internalizing moieties may be modified tomake them more resistant to cleavage by proteases. For example, thestability of an internalizing moiety comprising a polypeptide may beincreased by substituting one or more of the naturally occurring aminoacids in the (L) configuration with D-amino acids. In variousembodiments, at least 1%, 5%, 10%, 20%, 50%, 80%, 90% or 100% of theamino acid residues of an internalizing moiety may be of the Dconfiguration. The switch from L to D amino acids neutralizes thedigestion capabilities of many of the ubiquitous peptidases found in thedigestive tract. Alternatively, enhanced stability of an internalizingmoiety comprising a peptide bond may be achieved by the introduction ofmodifications of the traditional peptide linkages. For example, theintroduction of a cyclic ring within the polypeptide backbone may conferenhanced stability in order to circumvent the effect of many proteolyticenzymes known to digest polypeptides in the stomach or other digestiveorgans and in serum. In still other embodiments, enhanced stability ofan internalizing moiety may be achieved by intercalating one or moredextrorotatory amino acids (such as, dextrorotatory phenylalanine ordextrorotatory tryptophan) between the amino acids of an internalizingmoiety. In exemplary embodiments, such modifications increase theprotease resistance of an internalizing moiety without affecting theactivity or specificity of the interaction with a desired targetmolecule.

(b) Homing Peptides

In certain aspects, an internalizing moiety may comprise a homingpeptide which selectively directs the subject chimeric MTM1 polypeptideacross a cellular membrane and into cells. In certain embodiment, aninternalizing moiety may comprise a homing peptide which selectivelydirects the subject chimeric MTM1 polypeptide to a target tissue (e.g.,muscle). For example, delivering a chimeric polypeptide to the musclecan be mediated by a homing peptide comprising an amino acid sequence ofASSLNIA (SEQ ID NO: 12). Further exemplary homing peptides are disclosedin WO 98/53804, which is incorporated by reference in its entirety.Additional examples of homing peptides include the HIV transactivator oftranscription (TAT) which comprises the nuclear localization sequenceTat48-60; Drosophila antennapedia transcription factor homeodomain(e.g., Penetratin which comprises Antp43-58 homeodomain 3rd helix);Homo-rginine peptides (e.g., Arg7 peptide-PKC-ε agonist protection ofischemic rat heart (“Arg7” disclosed as SEQ ID NO: 13)); alpha-helicalpeptides; cationic peptides (“superpositively” charged proteins).

Additionally, homing peptides for a target tissue (or organ) can beidentified using various methods well known in the art. Once identified,a homing peptide that is selective for a particular target tissue can beused, in certain embodiments.

An exemplary method is the in vivo phage display method. Specifically,random peptide sequences are expressed as fusion peptides with thesurface proteins of phage, and this library of random peptides areinfused into the systemic circulation. After infusion into host mice,target tissues or organs are harvested, the phage is then isolated andexpanded, and the injection procedure repeated two more times. Eachround of injection includes, by default, a negative selection component,as the injected virus has the opportunity to either randomly bind totissues, or to specifically bind to non-target tissues. Virus sequencesthat specifically bind to non-target tissues will be quickly eliminatedby the selection process, while the number of non-specific binding phagediminishes with each round of selection. Many laboratories haveidentified the homing peptides that are selective for vasculature ofbrain, kidney, lung, skin, pancreas, intestine, uterus, adrenal gland,retina, muscle, prostate, or tumors. See, for example, Samoylova et al.,1999, Muscle Nerve, 22:460; Pasqualini et al., 1996, Nature, 380:364;Koivunen et al., 1995, Biotechnology, 13:265; Pasqualini et al., 1995,J. Cell Biol., 130:1189; Pasqualini et al., 1996, Mole. Psych., 1:421,423; Rajotte et al., 1998, J. Clin. Invest., 102:430; Rajotte et al.,1999, J. Biol. Chem., 274:11593. See, also, U.S. Pat. Nos. 5,622,699;6,068,829; 6,174,687; 6,180,084; 6,232,287; 6,296,832; 6,303,573;6,306,365.

III. Chimeric Polypeptides

Chimeric polypeptides of the present invention can be made in variousmanners. In certain embodiments, the C-terminus of an MTM1 polypeptidecan be linked to the N-terminus of an internalizing moiety (e.g., anantibody or a homing peptide). Alternatively, the C-terminus of aninternalizing moiety (e.g., an antibody or a homing peptide) can belinked to the N-terminus of an MTM1 polypeptide. For example, chimericpolypeptides can be designed to place the MTM1 polypeptide at the aminoor carboxy terminus of either the antibody heavy or light chain of mAb3E10. In certain embodiments, potential configurations include the useof truncated portions of an antibody's heavy and light chain sequences(e.g., mAB 3E10) as needed to maintain the functional integrity of theattached MTM1 polypeptide. Further still, the internalizing moiety canbe linked to an exposed internal (non-terminus) residue of MTM1 or avariant thereof. In further embodiments, any combination of theMTM1-internalizing moiety configurations can be employed, therebyresulting in an MTM1:internalizing moiety ratio that is greater than 1:1(e.g., two MTM1 molecules to one internalizing moiety).

The MTM1 polypeptide and the internalizing moiety may be conjugateddirectly to each other. Alternatively, they may be linked to each othervia a linker sequence, which separates the MTM1 polypeptide and theinternalizing moiety by a distance sufficient to ensure that each domainproperly folds into its secondary and tertiary structures. Preferredlinker sequences (1) should adopt a flexible extended conformation, (2)should not exhibit a propensity for developing an ordered secondarystructure which could interact with the functional domains of the MTM1polypeptide or the internalizing moiety, and (3) should have minimalhydrophobic or charged character, which could promote interaction withthe functional protein domains. Typical surface amino acids in flexibleprotein regions include Gly, Asn and Ser. Permutations of amino acidsequences containing Gly, Asn and Ser would be expected to satisfy theabove criteria for a linker sequence. Other near neutral amino acids,such as Thr and Ala, can also be used in the linker sequence. In aspecific embodiment, a linker sequence length of about 15 amino acidscan be used to provide a suitable separation of functional proteindomains, although longer or shorter linker sequences may also be used.The length of the linker sequence separating the MTM1 polypeptide andthe internalizing moiety can be from 5 to 500 amino acids in length, ormore preferably from 5 to 100 amino acids in length. Preferably, thelinker sequence is from about 5-30 amino acids in length. In preferredembodiments, the linker sequence is from about 5 to about 20 aminoacids, and is advantageously from about 10 to about 20 amino acids. Inother embodiments, the linker joining the MTM1 polypeptide to aninternalizing moiety can be a constant domain of an antibody (e.g.,constant domain of mAb 3E10 or all or a portion of an Fc region ofanother antibody). By way of example, the linker that joins MTM1 with aninternalizing moiety is GSTSGSGKSSEGKG (SEQ ID NO: 10). In certainembodiments, the linker is a cleavable linker. As noted above, thechimeric polypeptide may include more than one linker, such as a linkerjoining the internalizing moiety to the MTM polypeptide and a linkerjoining portions of the internalizing moiety to each other (e.g., alinker joining a VH and VL domain of a single chain Fv fragment). Whenthe chimeric polypeptide includes more than one linker, such as twolinkers, the linkers are independently selected and may be the same ordifferent.

In certain embodiments, the chimeric polypeptides of the presentinvention can be generated using well-known cross-linking reagents andprotocols. For example, there are a large number of chemicalcross-linking agents that are known to those skilled in the art anduseful for cross-linking the MTM1 polypeptide with an internalizingmoiety (e.g., an antibody). For example, the cross-linking agents areheterobifunctional cross-linkers, which can be used to link molecules ina stepwise manner. Heterobifunctional cross-linkers provide the abilityto design more specific coupling methods for conjugating proteins,thereby reducing the occurrences of unwanted side reactions such ashomo-protein polymers. A wide variety of heterobifunctionalcross-linkers are known in the art, including succinimidyl4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC),m-Maleimidobenzoyl-N-hydroxysuccinimide ester (MBS); N-succinimidyl(4-iodoacetyl)aminobenzoate (SIAB), succinimidyl 4-(p-maleimidophenyl)butyrate (SMPB), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimidehydrochloride (EDC);4-succinimidyloxycarbonyl-a-methyl-a-(2-pyridyldithio)-toluene (SMPT),N-succinimidyl 3-(2-pyridyldithio)propionate (SPDP), succinimidyl6-[3-(2-pyridyldithio)propionate]hexanoate (LC-SPDP). Thosecross-linking agents having N-hydroxysuccinimide moieties can beobtained as the N-hydroxysulfosuccinimide analogs, which generally havegreater water solubility. In addition, those cross-linking agents havingdisulfide bridges within the linking chain can be synthesized instead asthe alkyl derivatives so as to reduce the amount of linker cleavage invivo. In addition to the heterobifunctional cross-linkers, there existsa number of other cross-linking agents including homobifunctional andphotoreactive cross-linkers. Disuccinimidyl subcrate (DSS),bismaleimidohexane (BMH) and dimethylpimelimidate.2 HCl (DMP) areexamples of useful homobifunctional cross-linking agents, andbis-[B-(4-azidosalicylamido)ethyl]disulfide (BASED) andN-succinimidyl-6(4′-azido-2′-nitrophenylamino)hexanoate (SANPAH) areexamples of useful photoreactive cross-linkers for use in thisinvention. For a recent review of protein coupling techniques, see Meanset al. (1990) Bioconjugate Chemistry. 1:2-12, incorporated by referenceherein.

One particularly useful class of heterobifunctional cross-linkers,included above, contain the primary amine reactive group,N-hydroxysuccinimide (NHS), or its water soluble analogN-hydroxysulfosuccinimide (sulfo-NHS). Primary amines (lysine epsilongroups) at alkaline pH's are unprotonated and react by nucleophilicattack on NHS or sulfo-NHS esters. This reaction results in theformation of an amide bond, and release of NHS or sulfo-NHS as aby-product. Another reactive group useful as part of aheterobifunctional cross-linker is a thiol reactive group. Common thiolreactive groups include maleimides, halogens, and pyridyl disulfides.Maleimides react specifically with free sulfhydryls (cysteine residues)in minutes, under slightly acidic to neutral (pH 6.5-7.5) conditions.Halogens (iodoacetyl functions) react with —SH groups at physiologicalpH's. Both of these reactive groups result in the formation of stablethioether bonds. The third component of the heterobifunctionalcross-linker is the spacer arm or bridge. The bridge is the structurethat connects the two reactive ends. The most apparent attribute of thebridge is its effect on steric hindrance. In some instances, a longerbridge can more easily span the distance necessary to link two complexbiomolecules.

Preparing protein-conjugates using heterobifunctional reagents is atwo-step process involving the amine reaction and the sulfhydrylreaction. For the first step, the amine reaction, the protein chosenshould contain a primary amine. This can be lysine epsilon amines or aprimary alpha amine found at the N-terminus of most proteins. Theprotein should not contain free sulfhydryl groups. In cases where bothproteins to be conjugated contain free sulfhydryl groups, one proteincan be modified so that all sulfhydryls are blocked using for instance,N-ethylmaleimide (see Partis et al. (1983) J. Pro. Chem. 2:263,incorporated by reference herein). Ellman's Reagent can be used tocalculate the quantity of sulfhydryls in a particular protein (see forexample Ellman et al. (1958) Arch. Biochem. Biophys. 74:443 and Riddleset al. (1979) Anal. Biochem. 94:75, incorporated by reference herein).

In certain specific embodiments, chimeric polypeptides of the inventioncan be produced by using a universal carrier system. For example, anMTM1 polypeptide can be conjugated to a common carrier such as proteinA, poly-L-lysine, hex-histidine (SEQ ID NO: 14), and the like. Theconjugated carrier will then form a complex with an antibody which actsas an internalizing moiety. A small portion of the carrier molecule thatis responsible for binding immunoglobulin could be used as the carrier.

In certain embodiments, chimeric polypeptides of the invention can beproduced by using standard protein chemistry techniques such as thosedescribed in Bodansky, M. Principles of Peptide Synthesis, SpringerVerlag, Berlin (1993) and Grant G. A. (ed.), Synthetic Peptides: AUser's Guide, W. H. Freeman and Company, New York (1992). In addition,automated peptide synthesizers are commercially available (e.g.,Advanced ChemTech Model 396; Milligen/Biosearch 9600). In any of theforegoing methods of cross-linking for chemical conjugation of MTM1 toan internalizing moiety, a cleavable domain or cleavable linker can beused. Cleavage will allow separation of the internalizing moiety and theMTM1 polypeptide. For example, following penetration of a cell by achimeric polypeptide, cleavage of the cleavable linker would allowseparation of MTM1 from the internalizing moiety.

In certain embodiments, the chimeric polypeptides of the presentinvention can be generated as a fusion protein containing a MTM1polypeptide and an internalizing moiety (e.g., an antibody or a homingpeptide), expressed as one contiguous polypeptide chain. Such chimericpolypeptides are referred to herein as recombinantly conjugated. Inpreparing such fusion proteins, a fusion gene is constructed comprisingnucleic acids which encode an MTM1 polypeptide and an internalizingmoiety, and optionally, a peptide linker sequence to span the MTM1polypeptide and the internalizing moiety. The use of recombinant DNAtechniques to create a fusion gene, with the translational product beingthe desired fusion protein, is well known in the art. Both the codingsequence of a gene and its regulatory regions can be redesigned tochange the functional properties of the protein product, the amount ofprotein made, or the cell type in which the protein is produced. Thecoding sequence of a gene can be extensively altered—for example, byfusing part of it to the coding sequence of a different gene to producea novel hybrid gene that encodes a fusion protein. Examples of methodsfor producing fusion proteins are described in PCT applicationsPCT/US87/02968, PCT/US89/03587 and PCT/US90/07335, as well as Trauneckeret al. (1989) Nature 339:68, incorporated by reference herein.Essentially, the joining of various DNA fragments coding for differentpolypeptide sequences is performed in accordance with conventionaltechniques, employing blunt-ended or stagger-ended termini for ligation,restriction enzyme digestion to provide for appropriate termini, fillingin of cohesive ends as appropriate, alkaline phosphatase treatment toavoid undesirable joining, and enzymatic ligation. Alternatively, thefusion gene can be synthesized by conventional techniques includingautomated DNA synthesizers. In another method, PCR amplification of genefragments can be carried out using anchor primers which give rise tocomplementary overhangs between two consecutive gene fragments which cansubsequently be annealed to generate a chimeric gene sequence (see, forexample, Current Protocols in Molecular Biology, Eds. Ausubel et al.John Wiley & Sons: 1992). The chimeric polypeptides encoded by thefusion gene may be recombinantly produced using various expressionsystems as is well known in the art (also see below).

Recombinantly conjugated chimeric polypeptides include embodiments inwhich the MTM1 polypeptide is conjugated to the N-terminus or C-terminusof the internalizing moiety.

In some embodiments, the immunogenicity of the chimeric polypeptide maybe reduced by identifying a candidate T-cell epitope within a junctionregion spanning the chimeric polypeptide and changing an amino acidwithin the junction region as described in U.S. Patent Publication No.2003/0166877.

Chimeric polypeptides of the invention have any of a number of uses. Forexample, the chimeric polypeptides can be used to identify bindingpartners of MTM1, such as proteins to which MTM1 endogenously binds, orsubstrates for MTM1. Chimeric polypeptides can also be used to imageskeletal muscle cells, such as MTM1 deficient skeletal muscle cells, andto study the subcellular localization of MTM1 in wildtype or MTM1deficient skeletal muscle cells. Chimeric polypeptides can also be usedas part of a therapeutic method to replace MTM1 protein in deficientcells, in animals, or in human patients. Chimeric polypeptides can beused alone or as part of a therapeutic regimen for treating myotubularmyopathy.

IV. MTM1-Related Nucleic Acids and Expression

In certain embodiments, the present invention makes use of nucleic acidsfor producing an MTM1 polypeptide (including bioactive fragments,variants, and fusions thereof). In certain specific embodiments, thenucleic acids may further comprise DNA which encodes an internalizingmoiety (e.g., an antibody or a homing peptide) for making a recombinantchimeric protein of the invention. All these nucleic acids arecollectively referred to as MTM1 nucleic acids.

The nucleic acids may be single-stranded or double-stranded, DNA or RNAmolecules. In certain embodiments, the disclosure relates to isolated orrecombinant nucleic acid sequences that are at least 80%, 85%, 90%, 95%,97%, 98%, 99% or 100% identical to a region of an MTM1 nucleotidesequence (e.g., SEQ ID NOs: 5, 7, and 9). In further embodiments, theMTM1 nucleic acid sequences can be isolated, recombinant, and/or fusedwith a heterologous nucleotide sequence, or in a DNA library.

In certain embodiments, MTM1 nucleic acids also include nucleotidesequences that hybridize under highly stringent conditions to any of theabove-mentioned native MTM1 nucleotide sequence, or complement sequencesthereof. One of ordinary skill in the art will understand readily thatappropriate stringency conditions which promote DNA hybridization can bevaried. For example, one could perform the hybridization at 6.0× sodiumchloride/sodium citrate (SSC) at about 45° C., followed by a wash of2.0×SSC at 50° C. For example, the salt concentration in the wash stepcan be selected from a low stringency of about 2.0×SSC at 50° C. to ahigh stringency of about 0.2×SSC at 50° C. In addition, the temperaturein the wash step can be increased from low stringency conditions at roomtemperature, about 22° C., to high stringency conditions at about 65° C.Both temperature and salt may be varied, or temperature or saltconcentration may be held constant while the other variable is changed.In one embodiment, the invention provides nucleic acids which hybridizeunder low stringency conditions of 6×SSC at room temperature followed bya wash at 2×SSC at room temperature.

Isolated nucleic acids which differ from the native MTM1 nucleic acidsdue to degeneracy in the genetic code are also within the scope of theinvention. For example, a number of amino acids are designated by morethan one triplet. Codons that specify the same amino acid, or synonyms(for example, CAU and CAC are synonyms for histidine) may result in“silent” mutations which do not affect the amino acid sequence of theprotein. However, it is expected that DNA sequence polymorphisms that dolead to changes in the amino acid sequences of the subject proteins willexist among mammalian cells. One skilled in the art will appreciate thatthese variations in one or more nucleotides (up to about 3-5% of thenucleotides) of the nucleic acids encoding a particular protein mayexist among individuals of a given species due to natural allelicvariation. Any and all such nucleotide variations and resulting aminoacid polymorphisms are within the scope of this invention.

In certain embodiments, the recombinant MTM1 nucleic acids may beoperably linked to one or more regulatory nucleotide sequences in anexpression construct. Regulatory nucleotide sequences will generally beappropriate for a host cell used for expression. Numerous types ofappropriate expression vectors and suitable regulatory sequences areknown in the art for a variety of host cells. Typically, said one ormore regulatory nucleotide sequences may include, but are not limitedto, promoter sequences, leader or signal sequences, ribosomal bindingsites, transcriptional start and termination sequences, translationalstart and termination sequences, and enhancer or activator sequences.Constitutive or inducible promoters as known in the art are contemplatedby the invention. The promoters may be either naturally occurringpromoters, or hybrid promoters that combine elements of more than onepromoter. An expression construct may be present in a cell on anepisome, such as a plasmid, or the expression construct may be insertedin a chromosome. In a preferred embodiment, the expression vectorcontains a selectable marker gene to allow the selection of transformedhost cells. Selectable marker genes are well known in the art and willvary with the host cell used. In certain aspects, this invention relatesto an expression vector comprising a nucleotide sequence encoding anMTM1 polypeptide and operably linked to at least one regulatorysequence. Regulatory sequences are art-recognized and are selected todirect expression of the encoded polypeptide. Accordingly, the termregulatory sequence includes promoters, enhancers, and other expressioncontrol elements. Exemplary regulatory sequences are described inGoeddel; Gene Expression Technology: Methods in Enzymology, AcademicPress, San Diego, Calif. (1990). It should be understood that the designof the expression vector may depend on such factors as the choice of thehost cell to be transformed and/or the type of protein desired to beexpressed. Moreover, the vector's copy number, the ability to controlthat copy number and the expression of any other protein encoded by thevector, such as antibiotic markers, should also be considered.

This invention also pertains to a host cell transfected with arecombinant gene which encodes an MTM1 polypeptide, an internalizingmoiety, or a chimeric polypeptide of the invention. The host cell may beany prokaryotic or eukaryotic cell. For example, an MTM1 polypeptide ora chimeric polypeptide may be expressed in bacterial cells such as E.coli, insect cells (e.g., using a baculovirus expression system), yeast,or mammalian cells. Other suitable host cells are known to those skilledin the art.

The present invention further pertains to methods of producing an MTM1polypeptide, an internalizing moiety, and/or a chimeric polypeptide ofthe invention. For example, a host cell transfected with an expressionvector encoding an MTM1 polypeptide, an internalizing moiety, or achimeric polypeptide can be cultured under appropriate conditions toallow expression of the polypeptide to occur. The polypeptide may besecreted and isolated from a mixture of cells and medium containing thepolypeptides. Alternatively, the polypeptides may be retained in thecytoplasm or in a membrane fraction and the cells harvested, lysed andthe protein isolated. A cell culture includes host cells, media andother byproducts. Suitable media for cell culture are well known in theart. The polypeptides can be isolated from cell culture medium, hostcells, or both using techniques known in the art for purifying proteins,including ion-exchange chromatography, gel filtration chromatography,ultrafiltration, electrophoresis, and immunoaffinity purification withantibodies specific for particular epitopes of the polypeptides (e.g.,an MTM1 polypeptide). In a preferred embodiment, the polypeptide is afusion protein containing a domain which facilitates its purification.

A recombinant MTM1 nucleic acid can be produced by ligating the clonedgene, or a portion thereof, into a vector suitable for expression ineither prokaryotic cells, eukaryotic cells (yeast, avian, insect ormammalian), or both. Expression vehicles for production of a recombinantpolypeptide include plasmids and other vectors. For instance, suitablevectors include plasmids of the types: pBR322-derived plasmids,pEMBL-derived plasmids, pEX-derived plasmids, pBTac-derived plasmids andpUC-derived plasmids for expression in prokaryotic cells, such as E.coli. The preferred mammalian expression vectors contain bothprokaryotic sequences to facilitate the propagation of the vector inbacteria, and one or more eukaryotic transcription units that areexpressed in eukaryotic cells. The pcDNAI/amp, pcDNAI/neo, pRc/CMV,pSV2gpt, pSV2neo, pSV2-dhfr, pTk2, pRSVneo, pMSG, pSVT7, pko-neo andpHyg derived vectors are examples of mammalian expression vectorssuitable for transfection of eukaryotic cells. Some of these vectors aremodified with sequences from bacterial plasmids, such as pBR322, tofacilitate replication and drug resistance selection in both prokaryoticand eukaryotic cells. Alternatively, derivatives of viruses such as thebovine papilloma virus (BPV-1), or Epstein-Barr virus (pHEBo,pREP-derived and p205) can be used for transient expression of proteinsin eukaryotic cells. The various methods employed in the preparation ofthe plasmids and transformation of host organisms are well known in theart. For other suitable expression systems for both prokaryotic andeukaryotic cells, as well as general recombinant procedures, seeMolecular Cloning A Laboratory Manual, 2nd Ed., ed. by Sambrook, Fritschand Maniatis (Cold Spring Harbor Laboratory Press, 1989) Chapters 16 and17. In some instances, it may be desirable to express the recombinantpolypeptide by the use of a baculovirus expression system. Examples ofsuch baculovirus expression systems include pVL-derived vectors (such aspVL1392, pVL1393 and pVL941), pAcUW-derived vectors (such as pAcUW1),and pBlueBac-derived vectors (such as the β-gal containing pBlueBacIII).

Techniques for making fusion genes are well known. Essentially, thejoining of various DNA fragments coding for different polypeptidesequences is performed in accordance with conventional techniques,employing blunt-ended or stagger-ended termini for ligation, restrictionenzyme digestion to provide for appropriate termini, filling-in ofcohesive ends as appropriate, alkaline phosphatase treatment to avoidundesirable joining, and enzymatic ligation. In another embodiment, thefusion gene can be synthesized by conventional techniques includingautomated DNA synthesizers. Alternatively, PCR amplification of genefragments can be carried out using anchor primers which give rise tocomplementary overhangs between two consecutive gene fragments which cansubsequently be annealed to generate a chimeric gene sequence (see, forexample, Current Protocols in Molecular Biology, eds. Ausubel et al.,John Wiley & Sons: 1992).

It should be understood that chimeric polypeptides can be made innumerous ways. For example, an MTM1 polypeptide and an internalizingmoiety can be made separately, such as recombinantly produced in twoseparate cell cultures from nucleic acid constructs encoding theirrespective proteins. Once made, the proteins can be chemicallyconjugated directly or via a linker. By way of another example, thechimeric polypeptide can be made as an inframe fusion in which theentire chimeric polypeptide, optionally including one or more linker, ismade from a nucleic acid construct that includes nucleotide sequenceencoding both the MTM1 polypeptide and the internalizing moiety.

V. Methods of Treatment

In certain embodiments, the present invention provides methods oftreating conditions associated with deficient or non-functionalmyotubularin 1 (MTM1) protein, such as myotubular myopathy. Thesemethods involve administering to an individual in need thereof atherapeutically effective amount of a chimeric polypeptide as describedabove. Specifically, the method comprises administering a chimericpolypeptide comprising (a) a myotubularin (MTM1) polypeptide orbioactive fragment thereof and (b) an internalizing moiety. Thesemethods are particularly aimed at therapeutic and prophylactictreatments of animals, and more particularly, humans.

MTM is a rare and severe X-linked muscle disorder that occurs with anestimated incidence of 1 male in every 50,000 births and is caused by adeficiency of MTM1, a phosphoinositide phosphatase (Bello A B et al.,Human Molecular Genetics, 2008, Vol. 17, No. 14). At birth MTM patientspresent with severe hypotonia and respiratory distress and those thatsurvive the neonatal period are often totally or partially dependentupon ventilator support (Taylor G S et al., Proc Natl Acad Sci USA. 2000August 1; 97(16):8910-5; Bello A B et al., Proc Natl Acad Sci USA. 2002November 12; 99(23):15060-5; Pierson C R et al., Neuromuscul Disord.2007 July; 17(7): 562-568; Herman G E et al., THE JOURNAL OF PEDIATRICSVOLUME 134, NUMBER 2). Patients with MTM exhibit delayed motormilestones and are susceptible to complications such as scoliosis,malocclusion, pyloric stenosis, spherocytosis, and gall and kidneystones, yet linear growth and intelligence are normal and the diseasefollows a non-progressive course (Herman G E et al., THE JOURNAL OFPEDIATRICS VOLUME 134, NUMBER 2). An additional complication is that MTMpatients are particularly susceptible to severe and even lifethreatening respiratory infections. Without being bound by theory, theserespiratory infections may be due to the decreased ability ofindividuals to produce and clear mucous, as well as weakening of lungtissue brought about by long term ventilator use. The average hospitalstay for neonatal MTM patients is ˜90 days, and the need for long-termventilatory assistance and in-home care, as well as the costs associatedwith medical complications impose a substantial personal and economicburden to patients and families.

MTMs are a family of related proteins that exhibit phosphoinositidephosphatase activity or alternatively bind phosphoinositides but arecatalytically inactive. MTM1, as well as other related MTM proteins(MTMRs) assemble individually or in heterodimers on endocytic vesiclesat various stages of subcellular transport. MTM1 associates with MTMR12and interacts with other endosomal proteins such the GTPase Rab5 and thePI 3-kinase Vps34 via the Vps15 adapter molecule. The differentialrecruitment and opposing activities of MTM1 PIP3 phosphatase and Vsp34PI-3 kinase likely coordinate the temporal membrane distribution of PIand PIP3 that directs the intracellular traffic patterns of endocyticvesicles. Although other MTM-related proteins possess PIP3 phosphataseactivity, their subcellular localization is sufficiently non-overlappingfrom that of MTM1 that they are unable to functionally compensate forthe absence of MTM1. MTM1 is ubiquitously expressed yet the absence ofMTM1 in skeletal muscle solely accounts for the pathophysiology of MTM(Blondeau F et al., Hum Mol. Genet. 2000 September 22; 9(15):2223-9;Taylor G S et al., Proc Natl Acad Sci USA. 2000 August 1; 97(16):8910-5;Bello A B et al., Proc Natl Acad Sci USA. 2002 November 12;99(23):15060-5), and suggests that the PIP3 phosphatase activity of MTM1possesses a unique subcellular function that is particularly crucial tonormal skeletal muscle function. MTM1 is expected to participate in themaintenance of the longitudinal and transverse architecture of theT-tubule system, and thus defects in the organization of thesestructures would impair excitation-contraction coupling, and result inthe ensuing muscle weakness and atrophy (Bello A B et al., HumanMolecular Genetics, 2008, Vol. 17, No. 14; Laporte J et al., HUMANMUTATION 15:393.409 (2000); Herman G E et al., THE JOURNAL OF PEDIATRICSVOLUME 134, NUMBER 2).

Given that the identity of particular endosomal compartments may consistas a defined ratio and distribution of PI and its phosphorylated forms,a therapeutic approach that either blocks PI-3 kinase and/oralternatively increases PIP4 (also “PI(4)P”), PIP5 (also “PI(5)P”) orPIP4,5 (also “PI(4,5)P₂”) is not likely to impart any therapeuticspecificity towards MTM. MTM1, MTMR1 and MTMR2 are the most closelyrelated phosphoinositide phosphatases and are expressed in skeletalmuscle, and suggests that pharmacologic upregulation of other MTMRscould provide a compensatory benefit to MTM. However, the subcellularlocations of MTMR1, MTMR2 and MTM1 do not sufficiently overlap (LorenzoO et al., Journal of Cell Science 119, 2953-2959 2005) and the mutationof MTMR2 in the recessive motor and sensory demyelinating neuropathyCharcot-Marie-Tooth type 4B (CMT4B) presents with pathological andclinical manifestations that are very different to those of MTM.Therefore, compensatory upregulation of MTMR2 or other MTM-relatedproteins is likely to provide little, if any, therapeutic compensationfor MTM1 deficiency. The mRNA rescue technologies based upon stop codonread-through may be effective for ˜20% of MTM patients yet there is noindication that technologies based upon exon skipping will be useful forthe ˜50% patients possessing deletions and splice site mutations(Laporte J et al., HUMAN MUTATION 15:393.409 (2000)). An approach basedupon IGF administration, myostatin inhibition or AKT activation wouldnot correct the underlying biochemical defect of MTM but couldcounteract any hypotrophic signaling that may exist in MTM.

An approach that restores MTM1 to skeletal muscle either through gene,stem cell or recombinant intravenous therapy is a desirable therapeuticstrategy for MTM. In certain embodiments, the present disclosureprovides chimeric polypeptides suitable for use in methods for treatingMTM. Exemplary chimeric polypeptides comprise (a) an MTM1 polypeptide ora bioactive fragment thereof and (b) an internalizing moiety. In certainembodiments, the internalizing moiety selectively targets the chimericpolypeptide to muscle cells and/or transits cellular membranes via theENT2 transporter.

Intravenous delivery of recombinant MTM1 may provide the greatestflexibility in dosing with the fewest logistical barriers todevelopment. For example, dosing of intravenous MTM1 can be titrated toeffect, or withdrawn if a particular patient experiences a side effect.

MTM 1 is a cytoplasmic enzyme and possesses no inherent muscleinternalizing moiety, therefore MTM1 may be conjugated to a cellpermeable protein to traverse the skeletal muscle sarcolemma and reachthe appropriate cytoplasmic compartments. Since MTM1 has been shown toretain PIP3 phosphatase activity following numerous genetic fusions suchas N and C-terminal genetic conjugation to purification tags such as GSTand 6-His (SEQ ID NO: 14) (Kim SA et al., J. Biol. Chem., Vol. 277,Issue 6, 4526-4531, Feb. 8, 2002), and fluorescent reporters such as redand green fluorescent protein (Chaussade C et al., MolecularEndocrinology 17 (12): 2448-2460 2003), MTM1 is expected to retainactivity following chemical and genetic conjugation to, e.g., Fv3E10, amuscle internalizing single chain antibody. Additionally, hMTM1maintains the ability to localize to early endosomes andimmunoprecipitate accessory proteins such as Vps15 and Vps34 followinggenetic conjugation to 6-His (SEQ ID NO: 14) and GST purification tags(Taylor GS et al., Proc Natl Acad Sci U S A. 2000 Aug. 1;97;(16):8910-5;Cao C et al., Traffic 2007; 8: 1052-1067; Kim SA et al., J. Biol. Chem.,Vol. 277, Issue 6, 4526-4531, Feb. 8, 2002), Green and Red FluorescentProteins (Cao C et al., Traffic 2007; 8: 1052-1067; Chaussade C et al.,Molecular Endocrinology 17 (12): 2448-2460 2003; Robinson FL et al.,Trends in Cell Biology, 2006, 16(8): 403-412), and flag epitope tagging(Cao C et al., Traffic 2007; 8: 1052-1067; Kim SA et al., J. Biol.Chem., Vol. 277, Issue 6, 4526-4531, Feb. 8, 2002). Therefore, chemicaland genetic conjugates of 3E10 and hMTM1 will retain the ability topenetrate cells, cleave PIP3 to PI, and associate with endosomalproteins.

The terms “treatment”, “treating”, and the like are used herein togenerally mean obtaining a desired pharmacologic and/or physiologiceffect. The effect may be prophylactic in terms of completely orpartially preventing a disease, condition, or symptoms thereof, and/ormay be therapeutic in terms of a partial or complete cure for a diseaseor condition and/or adverse effect attributable to the disease orcondition. “Treatment” as used herein covers any treatment of a diseaseor condition of a mammal, particularly a human, and includes: (a)preventing the disease or condition from occurring in a subject whichmay be predisposed to the disease or condition but has not yet beendiagnosed as having it; (b) inhibiting the disease or condition (e.g.,arresting its development); or (c) relieving the disease or condition(e.g., causing regression of the disease or condition, providingimprovement in one or more symptoms). For example, “treatment” of MTMencompasses a complete reversal or cure of the disease, or any range ofimprovement in conditions and/or adverse effects attributable to MTM.Merely to illustrate, “treatment” of MTM includes an improvement in anyof the following effects associated with MTM or combination thereof:short life expectancy, respiratory insufficiency (partially orcompletely), poor muscle tone, drooping eyelids, poor strength inproximal muscles, poor strength in distal muscles, facial weakness withor without eye muscle weakness, abnormal curvature of the spine, jointdeformities, and weakness in the muscles that control eye movement(ophthalmoplegia). Improvements in any of these conditions can bereadily assessed according to standard methods and techniques known inthe art. The population of subjects treated by the method of the diseaseincludes subjects suffering from the undesirable condition or disease,as well as subjects at risk for development of the condition or disease.

By the term “therapeutically effective dose” or “effective amount” ismeant a dose that produces the desired effect for which it isadministered. The exact dose will depend on the purpose of thetreatment, and will be ascertainable by one skilled in the art usingknown techniques (see, e.g., Lloyd (1999) The Art, Science andTechnology of Pharmaceutical Compounding).

In certain embodiments, one or more chimeric polypeptides of the presentinvention can be administered, together (simultaneously) or at differenttimes (sequentially). In addition, chimeric polypeptides of the presentinvention can be administered in combination with one or more additionalcompounds or therapies for treating myotubular myopathy or for treatingneuromuscular disorders in general. For example, one or more chimericpolypeptides can be co-administered in conjunction with one or moretherapeutic compounds. The combination therapy may encompasssimultaneous or alternating administration. In addition, the combinationmay encompass acute or chronic administration. Optionally, the chimericpolypeptide of the present invention and additional compounds act in anadditive or synergistic manner for treating myotubular myopathy. By wayof example, the present method may be used in combination with any ofthe MTM therapeutic methods as described above (e.g., compensatoryupregulation of MTMR2 or other MTM-related proteins, or mRNA rescuetechnologies based upon stop codon read-through) to achieve an additiveor synergistic effect. Additional compounds to be used in combinationtherapies include, but are not limited to, small molecules,polypeptides, antibodies, antisense oligonucleotides, and siRNAmolecules. Further, combination therapy also includes the methodsdisclosed herein together with other therapies for MTM (e.g., physicaltherapy, ventilatory support, occupational therapy, accupuncture, etc.).Depending on the nature of the combinatory therapy, administration ofthe chimeric polypeptides of the invention may be continued while theother therapy is being administered and/or thereafter. Administration ofthe chimeric polypeptides may be made in a single dose, or in multipledoses. In some instances, administration of the chimeric polypeptides iscommenced at least several days prior to the other therapy, while inother instances, administration is begun either immediately before or atthe time of the administration of the other therapy.

Regardless of whether the chimeric polypeptide is adminstered as a soleor conjoint therapy, methods of treating including administering asingle dose or multiple doses. Multiple doses include administering thechimeric polypeptide at specified intervals, such as daily, weekly,twice monthly, monthly, etc. Multiple doses include an administrationscheme in which chimeric polypeptide is administered at specifiedintervals for the life of the patient.

VI. Gene Therapy

Conventional viral and non-viral based gene transfer methods can be usedto introduce nucleic acids encoding polypeptides of MTM1 in mammaliancells or target tissues. Such methods can be used to administer nucleicacids encoding polypeptides of the invention (e.g., MTM1, includingvariants thereof) to cells in vitro. In some embodiments, the nucleicacids encoding MTM1 are administered for in vivo or ex vivo gene therapyuses. Non-viral vector delivery systems include DNA plasmids, nakednucleic acid, and nucleic acid complexed with a delivery vehicle such asa liposome. Viral vector delivery systems include DNA and RNA viruses,which have either episomal or integrated genomes after delivery to thecell. Such methods are well known in the art.

Methods of non-viral delivery of nucleic acids encoding engineeredpolypeptides of the invention include lipofection, microinjection,biolistics, virosomes, liposomes, immunoliposomes, polycation orlipid:nucleic acid conjugates, naked DNA, artificial virions, andagent-enhanced uptake of DNA. Lipofection methods and lipofectionreagents are well known in the art (e.g., Transfectam™ and Lipofectin™).Cationic and neutral lipids that are suitable for efficientreceptor-recognition lipofection of polynucleotides include those ofFelgner, WO 91/17424, WO 91/16024. Delivery can be to cells (ex vivoadministration) or target tissues (in vivo administration). Thepreparation of lipid:nucleic acid complexes, including targetedliposomes such as immunolipid complexes, is well known to one of skillin the art.

The use of RNA or DNA viral based systems for the delivery of nucleicacids encoding MTM1 or its variants take advantage of highly evolvedprocesses for internalizing a virus to specific cells in the body andtrafficking the viral payload to the nucleus. Viral vectors can beadministered directly to patients (in vivo) or they can be used to treatcells in vitro and the modified cells are administered to patients (exvivo). Conventional viral based systems for the delivery of polypeptidesof the invention could include retroviral, lentivirus, adenoviral,adeno-associated and herpes simplex virus vectors for gene transfer.Viral vectors are currently the most efficient and versatile method ofgene transfer in target cells and tissues. Integration in the hostgenome is possible with the retrovirus, lentivirus, and adeno-associatedvirus gene transfer methods, often resulting in long term expression ofthe inserted transgene. Additionally, high transduction efficiencieshave been observed in many different cell types and target tissues.

The tropism of a retrovirus can be altered by incorporating foreignenvelope proteins, expanding the potential target population of targetcells. Lentiviral vectors are retroviral vectors that are able totransduce or infect non-dividing cells and typically produce high viraltiters. Selection of a retroviral gene transfer system would thereforedepend on the target tissue. Retroviral vectors are comprised ofcis-acting long terminal repeats with packaging capacity for up to 6-10kb of foreign sequence. The minimum cis-acting LTRs are sufficient forreplication and packaging of the vectors, which are then used tointegrate the therapeutic gene into the target cell to provide permanenttransgene expression. Widely used retroviral vectors include those basedupon murine leukemia virus (MuLV), gibbon ape leukemia virus (GaLV),Simian Immuno deficiency virus (SW), human immuno deficiency virus(HIV), and combinations thereof, all of which are well known in the art.

In applications where transient expression of the polypeptides of theinvention is preferred, adenoviral based systems are typically used.Adenoviral based vectors are capable of very high transductionefficiency in many cell types and do not require cell division. Withsuch vectors, high titer and levels of expression have been obtained.This vector can be produced in large quantities in a relatively simplesystem. Adeno-associated virus (“AAV”) vectors are also used totransduce cells with target nucleic acids, e.g., in the in vitroproduction of nucleic acids and peptides, and for in vivo and ex vivogene therapy procedures. Construction of recombinant AAV vectors aredescribed in a number of publications, including U.S. Pat. No.5,173,414; Tratschin et al., Mol. Cell. Biol. 5:3251-3260 (1985);Tratschin, et al.; Mol. Cell. Biol. 4:2072-2081 (1984); Hermonat &Muzyczka, PNAS 81:6466-6470 (1984); and Samulski et al., J. Virol.63:03822-3828 (1989).

Recombinant adeno-associated virus vectors (rAAV) are a promisingalternative gene delivery systems based on the defective andnonpathogenic parvovirus adeno-associated type 2 virus. All vectors arederived from a plasmid that retains only the AAV 145 bp invertedterminal repeats flanking the transgene expression cassette. Efficientgene transfer and stable transgene delivery due to integration into thegenomes of the transduced cell are key features for this vector system.

Replication-deficient recombinant adenoviral vectors (Ad) can beengineered such that a transgene replaces the Ad E1a, E1b, and E3 genes;subsequently the replication defector vector is propagated in human 293cells that supply deleted gene function in trans. Ad vectors cantransduce multiple types of tissues in vivo, including nondividing,differentiated cells such as those found in the liver, kidney and musclesystem tissues. Conventional Ad vectors have a large carrying capacity.

Packaging cells are used to form virus particles that are capable ofinfecting a host cell. Such cells include 293 cells, which packageadenovirus, and 42 cells or PA317 cells, which package retrovirus. Viralvectors used in gene therapy are usually generated by producer cell linethat packages a nucleic acid vector into a viral particle. The vectorstypically contain the minimal viral sequences required for packaging andsubsequent integration into a host, other viral sequences being replacedby an expression cassette for the protein to be expressed. The missingviral functions are supplied in trans by the packaging cell line. Forexample, AAV vectors used in gene therapy typically only possess ITRsequences from the AAV genome which are required for packaging andintegration into the host genome. Viral DNA is packaged in a cell line,which contains a helper plasmid encoding the other AAV genes, namely repand cap, but lacking ITR sequences. The cell line is also infected withadenovirus as a helper. The helper virus promotes replication of the AAVvector and expression of AAV genes from the helper plasmid. The helperplasmid is not packaged in significant amounts due to a lack of ITRsequences. Contamination with adenovirus can be reduced by, e.g., heattreatment to which adenovirus is more sensitive than AAV.

In many gene therapy applications, it is desirable that the gene therapyvector be delivered with a high degree of specificity to a particulartissue type. A viral vector is typically modified to have specificityfor a given cell type by expressing a ligand as a fusion protein with aviral coat protein on the viruses outer surface. The ligand is chosen tohave affinity for a receptor known to be present on the cell type ofinterest. This principle can be extended to other pairs of virusexpressing a ligand fusion protein and target cell expressing areceptor. For example, filamentous phage can be engineered to displayantibody fragments (e.g., FAB or Fv) having specific binding affinityfor virtually any chosen cellular receptor. Although the abovedescription applies primarily to viral vectors, the same principles canbe applied to nonviral vectors. Such vectors can be engineered tocontain specific uptake sequences thought to favor uptake by specifictarget cells, such as muscle cells.

Gene therapy vectors can be delivered in vivo by administration to anindividual patient, by systemic administration (e.g., intravenous,intraperitoneal, intramuscular, subdermal, or intracranial infusion) ortopical application. Alternatively, vectors can be delivered to cells exvivo, such as cells explanted from an individual patient (e.g.,lymphocytes, bone marrow aspirates, tissue biopsy) or universal donorhematopoietic stem cells, followed by reimplantation of the cells into apatient, usually after selection for cells which have incorporated thevector.

Ex vivo cell transfection for diagnostics, research, or for gene therapy(e.g., via re-infusion of the transfected cells into the host organism)is well known to those of skill in the art. For example, cells areisolated from the subject organism, transfected with a nucleic acid(gene or cDNA) encoding, e.g., MTM1 or its variants, and re-infused backinto the subject organism (e.g., patient). Various cell types suitablefor ex vivo transfection are well known to those of skill in the art.

In certain embodiments, stem cells are used in ex vivo procedures forcell transfection and gene therapy. The advantage to using stem cells isthat they can be differentiated into other cell types in vitro, or canbe introduced into a mammal (such as the donor of the cells) where theywill engraft in the bone marrow. Stem cells are isolated fortransduction and differentiation using known methods.

Vectors (e.g., retroviruses, adenoviruses, liposomes, etc.) containingtherapeutic nucleic acids can be also administered directly to theorganism for transduction of cells in vivo. Alternatively, naked DNA canbe administered. Administration is by any of the routes normally usedfor introducing a molecule into ultimate contact with blood or tissuecells. Suitable methods of administering such nucleic acids areavailable and well known to those of skill in the art, and, althoughmore than one route can be used to administer a particular composition,a particular route can often provide a more immediate and more effectivereaction than another route.

Pharmaceutically acceptable carriers are determined in part by theparticular composition being administered, as well as by the particularmethod used to administer the composition. Accordingly, there is a widevariety of suitable formulations of pharmaceutical compositions of thepresent invention, as described herein.

VII. Methods of Administration

Various delivery systems are known and can be used to administer thechimeric polypeptides of the invention, e.g., various formulations,encapsulation in liposomes, microparticles, microcapsules, recombinantcells capable of expressing the compound, receptor-mediated endocytosis(see, e.g., Wu and Wu, 1987, J. Biol. Chem. 262:4429-4432). Methods ofintroduction can be enteral or parenteral, including but not limited to,intradermal, transdermal, intramuscular, intraperitoneal, intravenous,subcutaneous, pulmonary, intranasal, intraocular, epidural, and oralroutes. In particular embodiments, parenteral introduction includesintramuscular, subcutaneous, intravenous, intravascular, andintrapericardial administration.

The chimeric polypeptides may be administered by any convenient route,for example, by infusion or bolus injection, by absorption throughepithelial or mucocutaneous linings (e.g., oral mucosa, rectal andintestinal mucosa, etc.) and may be administered together with otherbiologically active agents. Administration can be systemic or local.Pulmonary administration can also be employed, e.g., by use of aninhaler or nebulizer, and formulation with an aerosolizing agent.

In certain embodiments, it may be desirable to administer the chimericpolypeptides of the invention locally to the area in need of treatment(e.g., muscle); this may be achieved, for example, and not by way oflimitation, by local infusion during surgery, topical application, e.g.,by injection, by means of a catheter, or by means of an implant, theimplant being of a porous, non-porous, or gelatinous material, includingmembranes, such as sialastic membranes, fibers, or commercial skinsubstitutes.

In other embodiments, the chimeric polypeptides of the invention can bedelivered in a vesicle, in particular, a liposome (see Langer, 1990,Science 249:1527-1533). In yet another embodiment, the chimericpolypeptides of the invention can be delivered in a controlled releasesystem. In another embodiment, a pump may be used (see Langer, 1990,supra). In another embodiment, polymeric materials can be used (seeHoward et al., 1989, J. Neurosurg. 71:105). In certain specificembodiments, the chimeric polypeptides of the invention can be deliveredintravenously.

In certain embodiments, the chimeric polypeptides are administered byintravenous infusion. In certain embodiments, the chimeric polypeptidesare infused over a period of at least 10, at least 15, at least 20, orat least 30 minutes. In other embodiments, the chimeric polypeptides areinfused over a period of at least 60, 90, or 120 minutes. Regardless ofthe infusion period, the invention contemplates that each infusion ispart of an overall treatment plan where chimeric polypeptide isadministered according to a regular schedule (e.g., weekly, monthly,etc.).

VIII. Pharmaceutical Compositions

In certain embodiments, the subject chimeric polypeptides of the presentinvention are formulated with a pharmaceutically acceptable carrier. Oneor more chimeric polypeptides can be administered alone or as acomponent of a pharmaceutical formulation (composition). The chimericpolypeptides may be formulated for administration in any convenient wayfor use in human or veterinary medicine. Wetting agents, emulsifiers andlubricants, such as sodium lauryl sulfate and magnesium stearate, aswell as coloring agents, release agents, coating agents, sweetening,flavoring and perfuming agents, preservatives and antioxidants can alsobe present in the compositions.

Formulations of the subject chimeric polypeptides include those suitablefor oral, nasal, topical, parenteral, rectal, and/or intravaginaladministration. The formulations may conveniently be presented in unitdosage form and may be prepared by any methods well known in the art ofpharmacy. The amount of active ingredient which can be combined with acarrier material to produce a single dosage form will vary dependingupon the host being treated and the particular mode of administration.The amount of active ingredient which can be combined with a carriermaterial to produce a single dosage form will generally be that amountof the compound which produces a therapeutic effect.

In certain embodiments, methods of preparing these formulations orcompositions include combining another type of therapeutic agents and acarrier and, optionally, one or more accessory ingredients. In general,the formulations can be prepared with a liquid carrier, or a finelydivided solid carrier, or both, and then, if necessary, shaping theproduct.

Formulations for oral administration may be in the form of capsules,cachets, pills, tablets, lozenges (using a flavored basis, usuallysucrose and acacia or tragacanth), powders, granules, or as a solutionor a suspension in an aqueous or non-aqueous liquid, or as anoil-in-water or water-in-oil liquid emulsion, or as an elixir or syrup,or as pastilles (using an inert base, such as gelatin and glycerin, orsucrose and acacia) and/or as mouth washes and the like, each containinga predetermined amount of a subject polypeptide therapeutic agent as anactive ingredient. Suspensions, in addition to the active compounds, maycontain suspending agents such as ethoxylated isostearyl alcohols,polyoxyethylene sorbitol, and sorbitan esters, microcrystallinecellulose, aluminum metahydroxide, bentonite, agar-agar and tragacanth,and mixtures thereof.

In solid dosage forms for oral administration (capsules, tablets, pills,dragees, powders, granules, and the like), one or more polypeptidetherapeutic agents of the present invention may be mixed with one ormore pharmaceutically acceptable carriers, such as sodium citrate ordicalcium phosphate, and/or any of the following: (1) fillers orextenders, such as starches, lactose, sucrose, glucose, mannitol, and/orsilicic acid; (2) binders, such as, for example, carboxymethylcellulose,alginates, gelatin, polyvinyl pyrrolidone, sucrose, and/or acacia; (3)humectants, such as glycerol; (4) disintegrating agents, such asagar-agar, calcium carbonate, potato or tapioca starch, alginic acid,certain silicates, and sodium carbonate; (5) solution retarding agents,such as paraffin; (6) absorption accelerators, such as quaternaryammonium compounds; (7) wetting agents, such as, for example, cetylalcohol and glycerol monostearate; (8) absorbents, such as kaolin andbentonite clay; (9) lubricants, such a talc, calcium stearate, magnesiumstearate, solid polyethylene glycols, sodium lauryl sulfate, andmixtures thereof; and (10) coloring agents. In the case of capsules,tablets and pills, the pharmaceutical compositions may also comprisebuffering agents. Solid compositions of a similar type may also beemployed as fillers in soft and hard-filled gelatin capsules using suchexcipients as lactose or milk sugars, as well as high molecular weightpolyethylene glycols and the like. Liquid dosage forms for oraladministration include pharmaceutically acceptable emulsions,microemulsions, solutions, suspensions, syrups, and elixirs. In additionto the active ingredient, the liquid dosage forms may contain inertdiluents commonly used in the art, such as water or other solvents,solubilizing agents and emulsifiers, such as ethyl alcohol, isopropylalcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzylbenzoate, propylene glycol, 1,3-butylene glycol, oils (in particular,cottonseed, groundnut, corn, germ, olive, castor, and sesame oils),glycerol, tetrahydrofuryl alcohol, polyethylene glycols and fatty acidesters of sorbitan, and mixtures thereof. Besides inert diluents, theoral compositions can also include adjuvants such as wetting agents,emulsifying and suspending agents, sweetening, flavoring, coloring,perfuming, and preservative agents.

Pharmaceutical compositions suitable for parenteral administration maycomprise one or more chimeric polypeptides in combination with one ormore pharmaceutically acceptable sterile isotonic aqueous or nonaqueoussolutions, dispersions, suspensions or emulsions, or sterile powderswhich may be reconstituted into sterile injectable solutions ordispersions just prior to use, which may contain antioxidants, buffers,bacteriostats, solutes which render the formulation isotonic with theblood of the intended recipient or suspending or thickening agents.Examples of suitable aqueous and nonaqueous carriers which may beemployed in the pharmaceutical compositions of the invention includewater, ethanol, polyols (such as glycerol, propylene glycol,polyethylene glycol, and the like), and suitable mixtures thereof,vegetable oils, such as olive oil, and injectable organic esters, suchas ethyl oleate. Proper fluidity can be maintained, for example, by theuse of coating materials, such as lecithin, by the maintenance of therequired particle size in the case of dispersions, and by the use ofsurfactants.

These compositions may also contain adjuvants, such as preservatives,wetting agents, emulsifying agents and dispersing agents. Prevention ofthe action of microorganisms may be ensured by the inclusion of variousantibacterial and antifungal agents, for example, paraben,chlorobutanol, phenol sorbic acid, and the like. It may also bedesirable to include isotonic agents, such as sugars, sodium chloride,and the like into the compositions. In addition, prolonged absorption ofthe injectable pharmaceutical form may be brought about by the inclusionof agents which delay absorption, such as aluminum monostearate andgelatin.

Injectable depot forms are made by forming microencapsule matrices ofone or more polypeptide therapeutic agents in biodegradable polymerssuch as polylactide-polyglycolide. Depending on the ratio of drug topolymer, and the nature of the particular polymer employed, the rate ofdrug release can be controlled. Examples of other biodegradable polymersinclude poly(orthoesters) and poly(anhydrides). Depot injectableformulations are also prepared by entrapping the drug in liposomes ormicroemulsions which are compatible with body tissue.

In certain embodiments, the chimeric polypeptides of the presentinvention are formulated in accordance with routine procedures as apharmaceutical composition adapted for intravenous administration tohuman beings. Where necessary, the composition may also include asolubilizing agent and a local anesthetic such as lidocaine to ease painat the site of the injection. Where the composition is to beadministered by infusion, it can be dispensed with an infusion bottlecontaining sterile pharmaceutical grade water or saline. Where thecomposition is administered by injection, an ampoule of sterile waterfor injection or saline can be provided so that the ingredients may bemixed prior to administration.

The amount of the chimeric polypeptides of the invention which will beeffective in the treatment of a tissue-related condition or disease(e.g., myotubular myopathy) can be determined by standard clinicaltechniques. In addition, in vitro assays may optionally be employed tohelp identify optimal dosage ranges. The precise dose to be employed inthe formulation will also depend on the route of administration, and theseriousness of the condition, and should be decided according to thejudgment of the practitioner and each subject's circumstances. However,suitable dosage ranges for intravenous administration are generallyabout 20-5000 micrograms of the active chimeric polypeptide per kilogrambody weight. Suitable dosage ranges for intranasal administration aregenerally about 0.01 pg/kg body weight to 1 mg/kg body weight. Effectivedoses may be extrapolated from dose-response curves derived from invitro or animal model test systems.

In certain embodiments, chimeric polypeptides and compositions of thedisclosure, including pharmaceutical preparations, are non-pyrogenic. Inother words, in certain embodiments, the compositions are substantiallypyrogen free. In one embodiment the formulations of the invention arepyrogen-free formulations which are substantially free of endotoxinsand/or related pyrogenic substances. Endotoxins include toxins that areconfined inside a microorganism and are released only when themicroorganisms are broken down or die. Pyrogenic substances also includefever-inducing, thermostable substances (glycoproteins) from the outermembrane of bacteria and other microorganisms. Both of these substancescan cause fever, hypotension and shock if administered to humans. Due tothe potential harmful effects, even low amounts of endotoxins must beremoved from intravenously administered pharmaceutical drug solutions.The Food & Drug Administration (“FDA”) has set an upper limit of 5endotoxin units (EU) per dose per kilogram body weight in a single onehour period for intravenous drug applications (The United StatesPharmacopeial Convention, Pharmacopeial Forum 26 (1):223 (2000)). Whentherapeutic proteins are administered in relatively large dosages and/orover an extended period of time (e.g., such as for the patient's entirelife), even small amounts of harmful and dangerous endotoxin could bedangerous. In certain specific embodiments, the endotoxin and pyrogenlevels in the composition are less then 10 EU/mg, or less then 5 EU/mg,or less then 1 EU/mg, or less then 0.1 EU/mg, or less then 0.01 EU/mg,or less then 0.001 EU/mg.

The foregoing applies to any of the chimeric polypeptides, compositions,and methods described herein. The invention specifically contemplatesany combination of the features of such chimeric polypeptides,compositions, and methods (alone or in combination) with the featuresdescribed for the various pharmaceutical compositions and route ofadministration described in this section.

IX. Animal Models of MTM

Mice possessing a targeted inactivation of the MTM1 gene (MTM1 KO) areborn at a submendellian distribution but otherwise appear normal.However, within the first weeks of life MTM1 KO mice begin to losemuscle mass that rapidly progresses to respiratory collapse and death ata median age of 7 weeks (14 weeks maximum). Myofibers of MTM KO miceappear hypotrophic and vacuolated with centrally located nucleisurrounded by mitochondria and glycogen, yet there is very littlesarcolemma damage and no evidence of apoptosis or inflammation.Ultrastructurally, MTM1 protein appears at submembranous and vesicles ofthe cytoplasm; and the triads of the T-tubule system of skeletal muscle(Bello A B et al., Proc Natl Acad Sci USA. 2002 Nov. 12;99(23):15060-5). Since the deficiency of MTM1 in skeletal muscle solelyaccounts for the phenotype in MTM1 KO mice, the constructs disclosedherein may be assessed for therapeutic efficacy using the MTM1 KO mousemodel. Further, mice possessing a targeted partial inactivation of theMTM1 gene can also serve as a suitable model system for the presentinvention. Such mouse models are known in the art. For example, inMTM1δ4 mice, exon 4 is replaced by a loxP site and the Cre allele isabsent (Buj-Bello et al., 2002, PNAS 99(23):15060-15065).

Accordingly, in certain embodiments, the present disclosure contemplatesmethods of surveying improvements in disease phenotype using the MTM1constructs (e.g., the chimeric polypeptides comprising MTM1) disclosedherein in a mouse model of MTM. Studies in MTM1 deficient micedemonstrate the marked phenotypic differences between wild-type and MTM1deficient mice (see, e.g., Buj-Bello et al., 2002, PNAS99(23):15060-15065). For example, a clear divergence in weight gainbetween normal and MTM1 deficient mice can be seen at ˜3 weeks of age.(Bello A B et al., Proc Natl Acad Sci USA. 2002 Nov. 12; 99(23):15060-5)Also, hanging assessment tests indicate a dramatic difference in thehanging performance between MTM1 deficient mice and normal mice.Additionally, MTM1 deficient mice demonstrate a significantdeterioration in grip strength (e.g., forelimb grip) as compared tonormal mice. Further, compared to normal mice which manifest almost nofoot dragging, MTM1 deficient mice demonstrate increased foot draggingas determined by gait analysis. Detailed protocols for evaluating theeffect of chimeric polypeptides comprising MTM1 in this animal model aredescribed herein (Example 4).

As such, upon administration (e.g., intravenously) to the MTM1 deficientmice, the ability of the chemical and/or genetic conjugate of a chimericpolypeptide comprising MTM1 (for example, the 3E10-hMTM1 chimericpolypeptide outlined in the examples) to improve one or more symptoms inMTM1 deficient mice (e.g., increase body weight and lifespan, decreasefoot drag, improve forelimb grip strength, improve the ability of thetreated mice to support themselves against the force of gravity. Furtherexperiments can also assess any improvement in isometric contractionforce of selected skeletal muscles, increased myofiber cross-sectionalarea, reduced central nuclei, morphometry, light/fluorescencemicroscopy, spontaneous activity, ex vivo myography, and normalizedNADH-TR staining. The serum and tissue levels of 3E10-hMTM1, as well asthe development of any anti-3E10-hMTM1 antibodies will also be evaluatedusing immunological-based detection methods.

Moreover, once it is established that 3E10*MTM1 or MTM1 genetic fusion(e.g., 3E10-G53-hMTM1 or 3E10-GSTS-hMTM1) results in an improvement inphenotype, a complete pharmacokinetic study to determine the effectivedose, clearance rate, volume of distribution, and half-life of 3E10-MTM1can be determined. The pharmacokinetics of 3E10-MTM1 will likely followa multi-compartment model in which various tissues exhibit differentdegrees of clearance, and simple assessments of serum half-life will notprovide sufficient information to calculate a therapeutic dosing rate.Therefore, the calculation of a dose and dosing rate will ultimately bederived from empirical observations of the pharmacokinetics,pharmacodynamics, toxicology of a given dose of 3E10-MTM1, and the rateand extent to which an increase in weight, wire-hang time, forelimb gripstrength, foot drag, spontaneous activity, myofiber diameter andlifespan, for example, are observed. The dose and dosing rate of3E10-MTM1 determined in a subsequent pharmacokinetic study can be theused as the standard comparator to evaluate optimized lots ofrecombinant 3E10-MTM1. The PK/PD/TK of the final product will then beexamined in larger animals such as rats, dogs, and primates.

The above mouse models provide a suitable animal model system forassessing the activity and effectiveness of the subject chimericpolypeptides. Further these models correlate strongly with MTM, andprovide an appropriate model for MTM. Activity of the polypeptide can beassessed in these mouse models, and the results compared to thatobserved in wildtype control animals and animals not treated with thechimeric polypeptides. The results can be evaluated by examining themice, as well as by examining the ultrastructure of their muscle cells.Similarly, the subject chimeric polypeptides can be evaluated usingcells in culture, for example, cells prepared from the mutant mice.

In other embodiments, a large animal model can also be used to assessthe activity and effectiveness of the subject chimeric polypeptides. Byway of example, a dog model may be a particularly useful system forstudying MTM. The affected dog carries a deficient MTM1 gene and,therefore, the studies described herein for a mouse model similarlyapply to a dog model. The evaluation dose of 3E10 chemically orgenetically conjugated to hMTM1 delivered to MTM1 deficient dogs will bedetermined empirically.

X. Other Suitable Models

Other suitable models for evaluating the activity of the chimericpolypeptides of the invention include cell free and cell based assays.Chimeric polypeptides will possess two properties: a bioactivity of MTM1and a cell penetrating activity of the internalizing moiety thatpromotes transfer across cell membrane and into cells. Suitable modelsallow evaluation of one or both of these activities.

By way of example, the chimeric polypeptides can be evaluated for cellpenetrating activity using primary cells in culture or a cell line. Thecells are contacted with the chimeric polypeptide and assayed todetermine whether and to what extent the chimeric polypeptide enteredthe cell. For example, IHC can be used to evaluate whether the chimericpolypeptide entered the cell, and the results compared to an appropriatecontrol. In certain embodiments in which transfer into the cell isthought to occur via the ENT2 transporter, cells can be examined in thepresence or absence of an ENT2 transporter inhibitor.

Further, as described herein, the chimeric polypeptides of the presentinvention can be used to test for functionality in cell-based assays. Inan exemplary embodiment, primary muscle cells from MTM1 deficient mice(e.g., MTM1δ4) can be used to determine whether treatment with thechimeric polypeptides described herein can improve the fusion deficiencyof myotubularin deficient myoblasts. Detailed methods are exemplifiedherein.

By way of further example, bioactivity of MTM1 can be measured in cellfree assays, for example, to confirm that the chimeric polypeptideretains the ability to bind suitable binding partners and/or retainsphosphatase activity. Similar assays can be conducted in primary cellsin culture or in a cell line in culture.

XI. Kits

In certain embodiments, the invention also provides a pharmaceuticalpackage or kit comprising one or more containers filled with at leastone chimeric polypeptide of the invention. Exemplary containers include,but are not limited to, vials, bottles, pre-filled syringes, IV bags,blister packs (comprising one or more pills). Optionally associated withsuch container(s) can be a notice in the form prescribed by agovernmental agency regulating the manufacture, use or sale ofpharmaceuticals or biological products, which notice reflects (a)approval by the agency of manufacture, use or sale for humanadministration, (b) directions for use, or both.

EXEMPLIFICATION

The invention now being generally described, it will be more readilyunderstood by reference to the following examples, which are includedmerely for purposes of illustration of certain aspects and embodimentsof the present invention, and are not intended to limit the invention.For example, the particular constructs and experimental design disclosedherein represent exemplary tools and methods for validating properfunction. As such, it will be readily apparent that any of the disclosedspecific constructs and experimental plan can be substituted within thescope of the present disclosure.

Example 1 Chemical Conjugation of 3E10 and hMTM1 (mAb3E10*hMTM1)

Chemical Conjugation

Monoclonal Ab 3E10 is of the IgG2a subtype and is derived from thefusion of spleen cells from an MRL/lpr/mpj mouse with FOX-NY hybridomacells (Mankodi A et al., Mol. Cell. 2002 July; 10(1):35-44). Tenmilligrams (10 mg) of mAb 3E10 is conjugated covalently to recombinanthuman MTM1 (AbNova) in a 1/1 molar ratio with the use of two differentheterobifunctional reagents, succinimidyl 3-(2-pyridyldithio)propionateand succinimidyl trans-4-(maleimidylmethyl)cyclo-hexane-1-carboxylate.This reaction modifies the lysine residues of mAb 3E10 into thiols andadds thiolreactive maleimide groups to MTM1 (Weisbart R H, et al., J.Immunol. 2000 June 1; 164(11):6020-6). After deprotection, the modifiedproteins are reacted to each other to create a stable thioether bond.Chemical conjugation is performed, and the products are fractionated bygel filtration chromatography. The composition of the fractions areassessed by native and SDS-PAGE in reducing and nonreducingenvironments. Fractions containing the greatest ratio of mAb 3E10*hMTM1chemical conjugate to free mAb 3E10 and free hMTM1 are pooled andselected for use in further studies.

Additional chemical conjugates are similarly made for later testing. Byway of non-limiting example: (a) hMTM1*3E10, (b) Fv3E10*hMTM1, (c)hMTM1*Fv3E10. Note that throughout the example, the abbreviation Fv isused to refer to a single chain Fv of 3E10. Similarly, mAb 3E10 and 3E10are used interchangeably. These and other chimeric polypeptides can betested for enzymatic activity and functionality using, for example, theassays detailed herein. Any chimeric polypeptide comprising an MTM1portion and an internalizing moiety can be similarly tested to confirmthat the chimeric polypeptide maintains the activity of MTM1 and thecell penetrating activity of the internalizing moiety. Reference to anyparticular chimeric polypeptide in these examples is merely for example.In certain embodiments, a chimeric polypeptide comprising the amino acidsequence set forth in SEQ ID NO: 11, or the amino acid sequence setforth in SEQ ID NO: 11 in the absence of one or both epitope tags istested in any one or more of these assays set forth in any of theexamples. In certain embodiments, a chimeric polypeptide in which theinternalizing moiety comprises an antibody or antigen-binding fragmentcomprising a light chain comprising the amino acid sequence of SEQ IDNO: 4 and comprising a heavy chain comprising the amino acid sequence ofSEQ ID NO: 2 is tested. In other embodiments, a chimeric polypeptide inwhich the internalizing moiety comprises an antibody or antigen-bindingfragment comprising a light chain comprising an amino acid sequence atleast 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 4 andcomprising a heavy chain comprising an amino acid sequence at least 95%,96%, 97%, 98%, or 99% identical to SEQ ID NO: 2 is tested.

In Vitro PI(3)P and PI(3,5)P Phosphatase Activity of mAb3E10*hMTM1

The phosphatase activity of equimolar dilutions encompassing 5 logsof 1) chemically conjugated mAb3E10*hMTM1, 2) the unconjugated mixtureof mAb 3E10 and hMTM1, 3) mAb 3E10 alone, or 4) hMTM1 alone areevaluated according to the instructions of the Malachite GreenPhosphatase Assay Kit (Echelon Biosciences, Inc.) and as described bySchaletzky, J. et al. (Current Biology 13:504-509, 2003; SupplementalData, S1-S3). Phosphatase activity is evaluated to determine whether thechemically conjugated 3E10*hMTM1 construct retains enzymatic activity.Phosphatase activity is evaluated in both a cell free system, as well asin BHK cells in culture (e.g., in BHK cells treated with the conjugatesand compared to BHK cells treated with controls). Evaluating phosphataseactivity in both cell free and cell-based systems is useful becauseassaying phosphatase activity in cells may be complicated by the factthat other proteins with PI(3)P and PI(3,5)P phosphatase activity exist(including numerous MTM1 related (MTMR) proteins having PI(3)P andPI(3,5)P phosphatase activity). Accordingly, and given that BHK cellsmay not provide a clean background for testing PI(3)P or PI(3,5)Pphosphatase activity of these constructs, activity of the constructswill be confirmed using both cell free and cell-based systems. Activitycriteria include, for example, demonstrated 50% activity of MTM1 alone(by weight) or demonstrated 50% activity of published levels (see, e.g.,Figure S1 in Supplemental Data by Schaletzky, J. et al.).

Although the present examples specifically use PIP3 (also known as“PI(3)P”) and PI(3,5)P as MTM1 substrates, one of skill would readilyunderstand that other phosphoinositide substrates as disclosed hereinmay also be used as a substrate for in vitro assays.

Cell Entry of mAb3E10*hMTM1 and Association with Endosomal Protein Vps34

-   1. BHK cells are transfected with the cDNA encoding hVps34 and/or    the cDNA for the ENT2 transporter that mediates cell uptake of mAb    3E10 (Hansen J E et al., J Biol. Chem. 2007 Jul. 20;    282(29):20790-3). Two days later (48 hours post transfection)    chemically conjugated mAb3E10*hMTM1 or a mixture of unconjugated mAb    3E10 and hMTM1 are applied to transfected cells, followed by    immunoprecipitations with anti-Vps34, anti-hMTM1, or anti-mAb3E10,    followed by immunoblot detection of mAb 3E10, hVps34, and hMTM1.    Reverse immunoprecipitations are performed as controls. BHK cells    are immunologically negative for human MTM1 (hMTM1), human Vps34    (hVps34) (Cao C et al., Traffic 2007; 8: 1052-1067). Cell entry of    mAb3E10*hMTM1 via the transfected ENT2 transporter will be verified    by treating transfected cells with an ENT2 inhibitor (NBMPR) prior    to addition of conjugated and unconjugated material.

Human MTM1 is known to immunoprecipitate hVps34 and hVps15 (Cao C etal., Traffic 2007; 8: 1052-1067), and immunoprecipitation of hVps15 maybe used as further validation of an endosomal association or as analternative to hVps34 for use in immunoprecipitations. Given that BHKcells are immunologically negative for hMTM1 and hVps34, any evidence ofco-immunoprecipitation of hVps34 and hMTM1, following addition ofconjugated, but not unconjugated material is consistent with theconclusion that hMTM1 was delivered to cells and retained the ability toassociate with hVps34.

-   -   Synthesis of human Vps34 and ENT2 cDNA: The cDNAs for hVps34 and        the ENT2 transporter are as previously published (Hansen J E et        al., J Biol. Chem. 2007 Jul. 20; 282(29):20790-3; Cao C et al.,        Traffic 2007; 8: 1052-1067). Each cDNA is cloned into a        CMV-based mammalian expression cassette and large scale preps        will be made using the Qiagen Mega Endo-free plasmid        purification kit.    -   Transfections: Ten micrograms of the plasmid pCMV hVps34 and/or        pCMV ENT2 are transfected into 80% confluent BHK cells using        commercially available transfection reagents. Forty-eight hours        after transfection chemically conjugated mAb3E10*hMTM1 or a        mixture of unconjugated mAb 3E10 and free hMTM1 is applied to        cells. Four to 6 hours later, the cells are washed, and treated        with saponin, which clears the cytoplasmic contents while        maintaining membrane structures intact (Cao C et al., Traffic        2007; 8: 1052-1067). To track the efficiency of transfection,        duplicate transfections are performed with plasmids encoding a        suitable reporter such as beta-galactosidase or GFP.    -   Immunoprecipitation and immunoblots: Immunoprecipitations are        carried out as previously described (Weisbart R H et al., Mol.        Immunol. 2003 March; 39(13):783-9; Cao C et al., Traffic 2007;        8: 1052-1067) with anti-hMTM1, anti-hVps34, and anti-mAb3E10        antibody, followed by immunoblot detection of        coimmunoprecipitated hMTM1, hVps34, and mAb3E10. Reverse        immunoprecipitations are also performed as controls. When        epitope tagging is not be employed, the presence of a coincident        anti-3E10 and anti-hMTM1 immunoreactive band of ˜190 kDa in        mAb3E10*hMTM1 treated cells versus 3E10-alone and hMTM1-alone        controls will constitute successful penetration of chemically        conjugated 3E10*hMTM1. If the mAb3E10*hMTM1 chemical conjugate        remains intact following cell penetration it should        immunoprecipitate transfected hVps34 and likewise hVps34 should        immunoprecipitate hMTM1 and 3E10. Tubulin detection is used as a        loading control.    -   ENT2 inhibition: To verify the specificity of mAb3E10*MTM1 for        the ENT2 transporter all groups will be treated with the ENT2        inhibitor nitrobenzylmercaptopurine riboside (NBMPR) for 1 hour        before addition of chemically conjugated mAb3E10*hMTM1 or a        mixture of free recombinant mAb 3E10 and free hMTM1, and 4 to 6        hours later the media and cells will be collected for        immunoprecipitations as described above.

-   2. Primary muscle cells are dissociated in parallel from Mtm1δ4    (and/or from other MTM1 deficient mice) and wildtype littermate mice    following established method. Briefly, skeletal muscles are    harvested, finely minced, and digested using dispase II and    collagenase D. Primary myogenic cells are cultured in growth medium    (GM: F10 20% FBS, 10 ng/ml 3/4-FGF, 1% pen/strep) on collagen-coated    plates and passaged when they reach approximately 70% confluency.

Cells are treated with recombinant Fv3E10-MTM1 at a range of doses andfixed for immunodetection, or cell lysates are collected at a timecourseof endpoints. When present, the myc or 6His-tags (SEQ ID NO: 14) of therecombinant Fv3E10-MTM1 protein, such as the protein set forth in SEQ IDNO: 11, is utilized for immunodetection and subsequent determination ofintracellular uptake and internal localization. Anti Fv3E10-MTM1 tagantibodies are also utilized to coimmunoprecipitate intracellularFv3E10-MTM1, to detect the presence of binding proteins, and to test theenzymatic activity (using the Malachite Green Phosphatase Assay Kitdescribed herein) of internally transported Fv3E10-MTM1 protein.

Correction of Fusion Deficit in Primary Muscle Cells

Primary muscle cells are dissociated in parallel from Mtm1δ4 (and/orfrom other MTM1 deficiency mouse), and wildtype littermate micefollowing established method. Briefly, skeletal muscles are harvested,finely minced, and digested using dispase II and collagenase D. Primarymyogenic cells are cultured in growth medium (GM: F10 20% FBS, 10 ng/ml3/4-FGF, 1% pen/strep) on collagen-coated plates and passaged when theyreach approximately 70% confluency. To assess for differences incellular proliferation, the same number of primary cells are plated forMtm1δ4 (and/or from other MTM1 deficient mice) and wild-type myoblasts.Cultures are followed daily for approximately 2 weeks, with or withoutrecombinant MTM1 or Fv3E10-MTM1 addition.

To determine whether treatment with recombinant protein improves the‘fusion’ deficiency of myotubularin deficient myoblasts, primary cellsare plated in a 12-well plate at a concentration of 2×10⁵ cells/well.Cells are maintained in differentiation medium consisting of DMEMsupplemented 2% horse serum for 7 days, with the medium changed daily.Formation of myotubes is monitored and documented by acquiringmicroscopic images daily. The fusion index in control andmyotubularin-deficient cultures of cells is calculated as the ratio offused nuclei within myotubes over the number of total nuclei. Fusionindices are compared in control, mutant, and treated cultures using aWilcoxon rank sum test. These assessments indicate whether treated cellsdiffer in content, proliferation and differentiation abilities.

Following testing or treatment, cultured FDB myofibers or differentiatedmyotubes are grown on chamber slides, fixed in methanol, blocked in 10%FBS/0.1% Triton X-100, and incubated with either mouse monoclonalantibodies against desmin or myosin heavy chain type 1, type 2a, or type2b, as described above. Positive fibers are counted and point-to-pointmeasurements of minferet diameter are made using a Nikon Eclipse 90imicroscope using NIS-Elements-AR software (Nikon, Melville, N.Y.).

Example 2 Genetic Construct of fv 3E10 and hMTM1 (Fv3E10-GS3-hMTM1 andFv3E10-GSTS-hMTM1)

Mammalian expression vectors encoding a genetic fusion of Fv3E10 andhMTM1 (fv3E10-GS3-hMTM1, comprising the scFv of mAb 3E10 fused to hMTM1by the GS3 linker - SEQ ID NO: 3; fv3E10-GSTS-hMTM1, comprising the scFvof mAb 3E10 fused to hMTM1 by the “GSTS” linker - SEQ ID NO: 10) aregenerated. An exemplary sequence for a fv3E10-GSTS-hMTM1 chimericpolypeptide is provided in SEQ ID NO: 11. Thus, chimeric polypeptides ofthe invention include a chimeric polypeptide comprising the amino acidsequence set forth in SEQ ID NO: 11, in the presence or absence of theepitope tags (e.g., SEQ ID NO: 11 contains a myc-tag internally and a6x-His tag at the C terminus (SEQ ID NO: 14)).

Following transfection, the cells are immunoprecipitated to verify thatthe genetic fusion retains the ability to associate with endosomalproteins, as described in Example 1. The conditioned media is alsoimmunoblotted to detect secretion of 3E10 and hMTM1 into the culturemedia. Following concentration of the conditioned media, phosphataseactivity against, e.g., PI(3)P or PI(3,5)P, is measured and theconcentrated material is applied to BHK cells expressing Vps34 and ENT2to further validate that the secreted hMTM1 fusion enters cells andretains the ability to associate with endosomal proteins. Note thatthese genetic fusions are also referred to as recombinant conjugates orrecombinantly produced conjugates.

Additional recombinantly produced conjugates will similarly be made forlater testing. By way of non-limiting example: (a) hMTM1-GS3-3E10, (b)3E10-GS3-hMTM1, (c) hMTM1-GS3-Fv3E10, (d) hMTM1-3E10, (e) 3E10-hMTM1,(f) hMTM1-Fv3E10. Note that throughout the example, the abbreviation Fvis used to refer to a single chain Fv of 3E10. Similarly, mAb 3E10 and3E10 are used interchangeably. These and other chimeric polypeptides canbe tested using, for example, the assays detailed herein.

-   -   Synthesis of cDNA for hMTM1 and Fv3E10: The cDNA for human MTM1        (accession number: NP 000243.1) and the cDNA for the mouse        Fv3E10 variable light chain linked to the 3E10 heavy chain along        with flanking restriction sites that facilitate cloning into        appropriate expression vectors are synthesized and sequenced. To        maximize expression, each cDNA will be codon optimized for        mammalian, pichia, and/or E. coli expression. The human MTM1        cDNA has three glycosylation consensus sequences that may become        modified upon secretion. The cDNA encoding an exemplary mouse        Fv3E10 variable light chain linked to the 3E10 heavy chain for        use herein contains a mutation that enhances the cell        penetrating capacity of the Fv fragment, which occurs at        position 31 (D31Q) of the full mouse 3E10 sequence (Zack D J et        al., J. Immunol. 1996 Sep. 1; 157(5):2082-8), and it is the        variant used in the examples. The resulting cDNAs will be cloned        into a mammalian expression cassette, or other appropriate        expression cassette, and large scale preps of the plasmid        pCMV-Fv3E10-G53-hMTM1 will be made using the Qiagen Mega        Endo-free plasmid purification kit. Exemplary sequences of mouse        3E10 VH and mouse 3E10 VL are depicted by SEQ ID NOs: 2 and 4,        respectively.    -   Transfections: Ten micrograms of the plasmid pCMV-hMTM1,        pCMV-Fv3E10-GS3-hMTM1, pCMV ENT2 or pCMV are transfected into        80% confluent BHK cells using commercially available        transfection reagents. Forty-eight hours after transfection, the        conditioned media is collected and concentrated, and the cells        are washed and treated with saponin as described in Example 1.        To track the efficiency of transfection duplicate transfections        are performed with plasmids encoding a suitable reporter such as        beta-galactosidase or GFP.    -   PIP3 phosphatase activity of genetically conjugated hMTM1        fusions: Cell lysates and concentrated conditioned media from        BHK cells transfected with pCMV-hMTM1, pCMV-hMTM1 fusion as        described herein or pCMV are collected and phosphatase activity,        e.g., against PI(3)P or PI(3,5)P from each transfection is        evaluated per the instructions of the Malachite Green        Phosphatase Assay Kit (Echelon Biosciences, Inc). The general        overview of experimental groups and expected results for certain        control groups are indicated in Table 1. The cells marked as “?”        indicate results to be obtained. Control groups 13-16 (see        Table 1) will include application of chemically conjugated        mAb3E10*MTM1 to pCMV (mock) transfected cells followed one day        later by assessment of phosphatase activity in cell lysates and        concentrated conditioned media. The following day (24 hours        later) phosphatase activity is examined in cell lysates and        concentrated conditioned media. For example, it is expected that        phosphatase activity will not be observed in the lysate of group        13 that have been pre-treated with ENT2 inhibitor (Group 13 in        Table 1). Additional control groups 17-18 will have a known        activity of chemically conjugated mAb3E10*MTM1 spiked into cell        lysates and concentrated conditioned media of pCMV (mock)        transfected cells and will serve as a positive control and        standard curve for phosphatase activity.

TABLE 1 Experimental design to evaluate PIP3 phosphatase activity,endosomal association and secretion of Fv3E10-GS3-MTM1 Table 1: Strategyto evaluate PIP3 phosphatase, endosomal association and secretion ofgenetically conjugated Fv3E10-GS3-MTM1 Is following PIP3 activity inprotein also immunoprecipitations? Transfected Immunoprecipitationimmunoprecipitated? Conditioned Group cDNAs antibody Vps34 hMTM1 3E10Lysate* media 1 pCMV hMTM1 + anti-hMTM1 Yes Yes No Yes No 2 CMV hVps34anti-hVps34 Yes Yes No Yes No 3 anti-3E10 No No No Yes No 4 pCMV hMTM1 +anti-hMTM1 No Yes No Yes No 5 CMV anti-hVps34 No No No Yes No 6anti-3E10 No No No Yes No 7 pCMV 3E10- anti-hMTM1 ? Yes Yes ? ? 8GS3-hMTM1 + anti-hVps34 Yes ? ? ? ? 9 CMV hVps34 anti-3E10 ? Yes Yes ? ?10 pCMV 3E10- anti-hMTM1 No Yes Yes ? ? 11 GS3-hMTM1 + anti-hVps34 No NoNo ? ? 12 CMV anti-3E10 No Yes Yes ? ? PIP3 activity in Application ofimmunoprecipitations? Transfected mAb3E10*hMTM1 to Conditioned GroupcDNAs ENT2 Inhibitor cells Lysate* media 13 CMV + CMV + + No Yes 14 ENT2− + Yes Yes 15 CMV + + ? Yes 16 − + ? Yes 17 CMV − − Yes, Spiked No 18 −− No Yes, Spiked *PIP3 phosphatase activities will be a function ofendogenous and hMTM1 dependent activity. Groups 13 through 16 arecontrols to shown that ENT2 controls the cell uptake of chemicalconjugates of 3E10*hMTM1, and Groups 17 and 18 are controls fordetection of MTM1 phosphatase activity in cell lysates and conditionedmedia

-   -   Cellular uptake of genetically conjugated hMTM1 fusions:        Chemically conjugated mAb3E10*hMTM1 and concentrated conditioned        media from BHK cells transfected with pCMV hMTM1 fusion or pCMV        is applied to BHK cells transfected 48 hours earlier with pCMV        ENT2 or pCMV Table 2. Immunoprecipitations will then proceed as        in Example 1. Treatment of duplicate groups with the NBMPR        transporter inhibitor will verify that uptake of 3E10 is        specific to the ENT2 transporter.

TABLE 2 Experimental design to evaluate if Fv3E10-GS3-hMTM1 enters cellsvia ENT2 and associates with endosomal proteins Table 2: Strategy toassess if genetically conjugated Fv3E10-GS3-hMTM1 enters cells via ENT2and associates with endosomal proteins Cells treated with Cells treatedwith SALINE before NBMPR before recombinant protein recombinant proteinIs following protein also Is following protein also cDNA Source of IPimmunoprecipitated? immunoprecipitated? Group Transfected 3E10-hMTM1Antibody Vps34 hMTM1 3E10 Vps34 hMTM1 3E10 1 pCMV 3E10 + hMTM1anti-hMTM1 No No No No No No 2 hVps34 + mixed anti-hVps34 Yes No No YesNo No 3 pCMV unconjugated anti-3E10 No No Yes No No No ENT2 4 pCMVanti-hMTM1 No No No No No No 5 hVps34 + anti-hVps34 Yes No No Yes No No6 pCMV anti-3E10 No No ? No No No 7 pCMV + anti-hMTM1 No No No No No No8 pCMV anti-hVps34 No No No No No No 9 ENT2 anti-3E10 No No Yes No No No10 pCMV mAb 3E10* anti-hMTM1 Yes* Yes Yes No No No 11 hVps34 + hMTM1anti-hVps34 Yes Yes* Yes* Yes No No 12 pCMV (chemically anti-3E10 Yes*Yes Yes No No No ENT2 conjugated) 13 pCMV anti-hMTM1 Yes* Yes Yes No NoNo 14 hVps34 + anti-hVps34 Yes Yes* Yes* Yes No No 15 pCMV anti-3E10Yes* Yes Yes No No No 16 pCMV + anti-hMTM1 No Yes Yes No No No 17 pCMVanti-hVps34 No No No No No No 18 ENT2 anti-3E10 No Yes Yes No No No 19pCMV Concentrated anti-hMTM1 ? ? ? No No No 20 hVps34 + conditionedanti-hVps34 Yes ? ? Yes No No 21 pCMV media anti-3E10 ? ? ? No No NoENT2 from pCMV 22 pCMV Fv3E10-GS3- anti-hMTM1 ? ? ? No No No 23 hVps34 +hMTM1 anti-hVps34 Yes ? ? Yes No No 24 pCMV transfection anti-3E10 ? ? ?No No No 25 pCMV + (genetically anti-hMTM1 No ? ? No No No 26 pCMVconjugated) anti-hVps34 No No No No No No 27 ENT2 anti-3E10 No ? ? No NoNo 28 pCMV Concentrated anti-hMTM1 Yes** Yes Yes Yes** Yes Yes 29Vps34 + conditioned anti-hVps34 Yes Yes** Yes** Yes Yes** Yes** 30 pCMVmedia anti-3E10 Yes** Yes Yes Yes** Yes Yes 3E10- from pCMV GS3- mockhMTM1 transfection *immunoprecipitated and detected by immunoblot onlyif immunoprecipitated in specific aim 1 table 2 groups 10 through 18.**assumes the genetic conjugate has no defects in association betweenVps34 and hMTM1.

Example 3 Recombinant Production of hMTM1 Fusions and Verification ofPIP3 Phosphatase Activity, Cell Penetration, and Endosomal Associationwith Vps34

Recombinant 3E10-GS3-hMTM1 or 3E10-GSTS-hMTM1 is produced using thePichia yeast protein expression system (Invitrogen, RCT). Pichiaexhibits excellent protein expression, high cell densities, controllableprocesses, generation stability, durability and product processing thatis similar to mammalian cells. Both secreted and nonsecreted forms of3E10-GS3-hMTM1 are produced. The hMTM1 sequence contains three potentialNXS/T glycosylation sites that may affect the biological activity of anysecreted material. In other embodiments, the chimeric polypeptides areproduced in a bacterial expression system (e.g., E. coli) or a mammaliancell expression system.

-   -   Construction of protein expression vectors for Pichia: Plasmid        construction, transfection, colony selection and culture of        Pichia will use kits and manuals per the manufacturer's        instructions (Invitrogen). The cDNAs for genetically conjugated        hMTM1 (3E10-GS3-hMTM1 or 3E10-GSTS-hMTM1) created and validated        in Example 2 are cloned into two alternative plasmids; PICZ for        intracellular expression and PICZalpha for secreted expression.        Protein expression from each plasmid is driven by the AOX1        promoter. Transfected Pichia are selected with Zeocin and        colonies are tested for expression of recombinant hMTM1 fusion.        High expressers will be selected and scaled for purification.    -   Purification of recombinant hMTM1 fusion: cDNA fusions with mAb        3E10 Fv are ligated into the yeast expression vector pPICZA        which is subsequently electroporated into the Pichia pastoris        X-33 strain. Colonies are selected with Zeocin (Invitrogen,        Carlsbad, Calif.) and identified with anti-his6 (SEQ ID NO: 14)        antibodies (Qiagen Inc, Valencia, Calif.). X-33 cells are grown        in baffled shaker flasks with buffered glycerol/methanol medium,        and protein synthesis is induced with 0.5% methanol according to        the manufacturer's protocol (EasySelect Pichia Expression Kit,        Invitrogen, Carlsbad, Calif.). The cells are lysed by two        passages through a French Cell Press at 20,000 lbs/in2, and        recombinant protein is purified from cell pellets solubilized in        9M guanidine HC1 and 2% NP40 by immobilized metal ion affinity        chromatography (IMAC) on Ni-NTA-Agarose (Qiagen, Valencia,        Calif.). Bound protein is eluted in 50 mM NaH2PO4 containing 300        mM NaC1, 500 mM imidazole, and 25% glycerol. Samples of eluted        fractions are electrophoresed in 4-20% gradient SDS-PAGE (NuSep        Ltd, Frenchs Forest, Australia), and recombinant proteins is        identified by Western blotting to nitrocellulose membranes        developed with cargo-specific mouse antibodies followed by        alkaline-phosphatase-conjugated goat antibodies to mouse IgG.        Alkaline phosphatase activity is measured by the chromogenic        substrate, nitroblue tetrazolium        chloride/5-bromo-4-chloro-3-indolylphosphate p-toluidine salt.        Proteins are identified in SDS-PAGE gels with GelCode Blue Stain        Reagent (Pierce Chemical Co., Rockford, Ill.). Eluted protein is        concentrated, reconstituted with fetal calf serum to 5%, and        exchange dialyzed 100-fold in 30,000 MWCO spin filters        (Millipore Corp., Billerica, Mass.) against McCoy's medium        (Mediatech, Inc., Herndon, Va.) containing 5% glycerol.    -   Expression is bacteria: Chimeric polypeptide may also be        expressed in a bacterial expression system, such as E. coli. In        such cases, nucleic acid construct encoding the chimeric        polypeptide is codon biased optimized for expression in E. coli.        For expression in E. coli, pGEX-2T GST expression vector is        used. Different strains of E. coli may be tested to optimize        expression, such as E. coli strains WCA, BL21(DE3), and        BL21(DE3) pLysE.    -   Quality assessment and formulation: Immunoblot against 3E10 and        hMTM1 will be used to verify the size and identity of        recombinant proteins, followed by silver staining to identify        the relative purity of among preparations of 3E10, MTM1 and        hMTM1 fusion. Recombinant material will be formulated in a        buffer and concentration (˜0.5 mg/ml) that is consistent with        the needs of subsequent in vivo administrations.    -   In vitro assessment of recombinant material: Based on studies        from Example 2, the amount of phosphatase activity (e.g.,        against PI(3)P or PI(3,5)P per mole of conjugate that exists in        the conditioned media of hMTM1 fusion transfected cells is        determined and this value is used as a standard for assessment        of phosphatase activity of pichia or E. coli-derived recombinant        hMTM1 fusion. As shown in Table 2, the relative PIP3 phosphatase        activity of chemically conjugated, mammalian cell-derived and        pichia-derived recombinant 3E10-GS3-hMTM1 on Vps34 transfected        BHK cells is similarly compared.

Example 4 In Vivo Assessment of Muscle Targeted hMTM1 in MTM KO Mice

Selection of a Mouse Model for Evaluation

Mice possessing a targeted inactivation of the MTM1 gene (MTM1 KO) areborn at a submendellian distribution but otherwise appear normal.However, within the first weeks of life MTM1 KO mice begin to losemuscle mass that rapidly progresses to respiratory collapse and death ata median age of 7 weeks (14 weeks maximum) (Bello A B et al., Proc NatlAcad Sci USA. 2002 Nov. 12; 99(23):15060-5). The progressive decline inMTM KO mice coincides with blunted weight gain, reduced forelimb gripstrength, reduced ability to support themselves against the force ofgravity and increased foot dragging (Bello A B et al., Proc Natl AcadSci USA. 2002 Nov. 12; 99(23):15060-5). Myofibers of MTM KO mice appearhypotrophic and vacuolated with centrally located nuclei surrounded bymitochondria and glycogen, yet there is very little sarcolemma damageand no evidence of apoptosis or inflammation (Bello A B et al., ProcNatl Acad Sci USA. 2002 Nov. 12; 99(23):15060-5). An advantage of usingMTM1 KO mice is the ability to use ELISA to track the serum and tissuelevels 3E10-GS3-MTM1, and will be necessary for the subsequentpharmacokinetic assessments and the respective pharmacodynamic andtoxicologic responses. To control whether a superphysiological level ofMTM1 is detrimental, 3E10-hMTM1 will be administered to wildtype MTM1homozygous mice (+/+).

Exemplary assays for assessing treated mice are described below. Inaddition, improvement in the condition of mice is assessed based on lifeexpectancy (increase in life expectancy in treated mice), weight, anddecrease in foot dragging.

Selection of Dose of 3E10-G53-hMTM1

The evaluation dose of 3E10 chemically or genetically conjugated tohMTM1 delivered to MTM KO mice is determined. As a starting point, todemonstrate a therapeutic response, a high dose (e.g., 5.0 mg/kg) isadministered intravenously twice per week for as long as 20 weeks ordeath, whichever comes first. A dose of 5 mg/kg of 3E10-GS3-MTM1 isdelivered twice per week to MTM KO mice for 20 weeks (Table 3), followedby assessment of changes in disease endpoints. Controls include vehicleand treated heterozygous MTM1 +/+ mice and vehicle treated MTM1 −/−mice. Development of anti-3E10-MTM1 antibodies is also monitored. If weestablish that intravenous 3E10*MTM1 or MTM1 genetic fusions (e.g.,3E10-GS3-hMTM1 or 3E10-GSTS-hMTM1) results in an improvement inphenotype, subsequent in vivo PK assessments in MTM KO mice will beinitiated to identify a dosing regimen that promotes the greatestpharmacodynamic effect with the least toxicologic consequences.

TABLE 3 In vivo dosing plan for chemically and genetically conjugated3E10-MTM1 Table 3: In vivo dosing plan for chemically and geneticallyconjugated 3E10-MTM1 Dose Age # of (mg/ Group Strain (weeks) miceTreatment kg) 1 MTM1 −/− 10 5 mAb 3E10*hMTM1 5 (chemically conjugated) 2MTM1 −/− 10 5 mAb 3E10 & hMTM1 5 (mixed unconjugated) 3 MTM1 −/− 10 5Fv3E10-GS3-hMTM1 5 (genetically conjugated) 4 MTM1 −/− 10 5 Vehicle NA 5MTM1 +/+ 10 5 mAb 3E10*hMTM1 5 (chemically conjugated) 6 MTM1 +/+ 10 5mAb 3E10 & hMTM1 5 (mixed unconjugated) 7 MTM1 +/+ 10 5 Fv3E10-GS3-hMTM15 (genetically conjugated) 8 MTM1 +/+ 10 5 Vehicle NA TimepointInformation: Dose twice per week for 20 weeks. Daily observations.Collect blood and tissues for IHC, H&E and protein isolation

-   -   Grip strength: The grip strength device (Columbus Instruments)        requires no training on behalf of the mouse. The whole body        tension test employed by others (Bello A B et al., Proc Natl        Acad Sci USA. 2002 Nov. 12; 99(23):15060-5) an animal tied by        its tail to a force transducer, the tail is then pinched, and        the tension exerted as the animal attempts to escape is        measured. Aside from the animal welfare implications of inducing        pain as a condition of a response, it is unclear how the pinch        force is standardized or how the tail is properly secured to the        transducer. As an acceptable alternative, a forelimb grip        strength test, normalized to mouse weight, would sufficiently        replicate the measurements of the whole body tension test, would        survive the scrutiny of an IACUC and can be performed with a        readily available force transducer (Columbus Instruments). The        angle at which the mouse is pulled from the metal grid will have        a proportional effect on the force measurement. Therefore, the        grip strength test is standardized by pulling the mouse by the        tail parallel to the horizontal grid and away from the force        transducer at a rate of about 5 cm/second. Four pulls separated        by ˜20 seconds of rest are used to gauge any fatigue.    -   Injection of chemically and genetically conjugated 3E10-MTM1.        3E10*hMTM1 or hMTM1 genetic fusion is formulated and diluted in        a buffer that is consistent with intravenous injection (e.g.        sterile saline solution or a buffered solution of 50 mM        Tris-HCl, pH 7.4, 0.15 M NaCl). The amount of 3E10*hMTM1 or        hMTM1 genetic fusion given to each mouse is calculated as        follows: dose (mg/kg)×mouse weight (kg)×stock concentration        (mg/ml)=volume (ml) of stock per mouse, q.s. to 100 ul with        vehicle.    -   Blood collection. Blood is collected by cardiac puncture at the        time that animals are sacrificed for tissue dissection. Serum is        removed and frozen at −80° C. To minimize the effects of thawing        and handling all analysis of 3E10*MTM1 or MTM1 genetic fusion        circulating in the blood is performed on the same day.    -   Tissue collection and preparation. Sampled tissues are divided        for immunoblot, formalin-fixed paraffin-embedded tissue blocks        and frozen sections in OCT. One half of the heart, liver, lung,        spleen, kidneys, quadriceps, EDL, soleus, diaphragm, and biceps        will be subdivided and frozen in plastic tubes for further        processing for immunoblot analysis. The remaining half of the        heart, liver, lung, spleen, kidneys, quadriceps, EDL, soleus,        diaphragm, and biceps will be subdivided, frozen in OCT tissue        sectioning medium, or fixed in zinc-formaldehyde fixation for 24        to 48 hours at 4° C. and paraffin embedded.    -   Histological evaluation. Brightfield microscopy of HE sections        are used to determine the percentage of centrally nucleated        myofibers and myofiber cross-sectional area from five randomly        selected fields. At least 200 fibers are counted per mouse per        muscle group. Other sections are stained for NADH-TR. Scoring of        blinded sections for central nuclei, myofiber cross-sectional        area, and normalization of NADH-TR staining is also performed.    -   Immunoblot. Protein isolation and immunoblot detection of 3E10        and MTM1 is performed as previously described (Weisbart R H et        al., Mol. Immunol. 2003 March; 39(13):783-9; Bello A B et al.,        Human Molecular Genetics, 2008, Vol. 17, No. 14; Lorenzo O et        al., Journal of Cell Science 119, 2953-2959 2005).    -   Analysis of circulating 3E10-hMTM1: An ELISA specific to human        3E10-MTM1 is developed and validated using commercially        available anti-human MTM1 antibodies. Recombinant 3E10-MTM1 is        diluted and used to generate a standard curve. Levels of        3E10-MTM1 are determined from dilutions of serum (normalized to        ng/ml of serum) or tissue extracts (normalized to ng/mg of        tissue).    -   Monitoring of anti-3E10-hMTM1 antibody responses. Purified        3E10-MTM1 used to inject MTM KO mice is plated onto high-binding        96 well ELISA plates at 1 ug/ml in coating buffer (Pierce        Biotech), allowed to coat overnight, blocked for 30 minutes in        1% nonfat drymilk (Biorad) in TBS, and rinsed three times in        TBS. Two-fold dilutions of sera from vehicle and 3E10-MTM1        injected animals are loaded into wells, allowed to incubate for        30 minutes at 37° C., washed three times, incubated with        horseradish peroxidase (HRP)-conjugated rabbit anti-mouse IgA,        IgG, and IgM, allowed to incubate for 30 minutes at 37° C., and        washed three times. Mouse anti-3E10-MTM1 antibodies are detected        with TMB liquid substrate and read at 405 nm in ELISA plate        reader. Polyclonal rabbit anti-mouse MTM1 (Bello A B et al.,        Human Molecular Genetics, 2008, Vol. 17, No. 14), followed by        HRP-conjugated goat anti-rabbit serves as the positive control        antibody reaction. Any absorbance at 405 nm greater than that of        vehicle treated MTM1 KO mice constitutes a positive        anti-3E10-MTM1 antibody response.    -   Statistical Analysis. Pairwise comparisons will employ Student's        t-test. Comparisons among multiple groups will employ ANOVA. In        both cases a p-value <0.05 will be considered statistically        significant.

The foregoing examples help illustrate the experiments that can be usedduring the making and testing of chimeric polypeptides for use in themethods described herein. Chimeric polypeptides comprising an MTM1portion and an internalizing moiety are tested using, for example, thesemethods. Any of the chimeric polypeptides described in the specificationcan be readily tested. Any chimeric polypeptide comprising an MTM1portion and an internalizing moiety can be similarly tested to confirmthat the chimeric polypeptide maintains the activity of MTM1 and thecell penetrating activity of the internalizing moiety. Reference to anyparticular chimeric polypeptide in these examples is merely for example.In certain embodiments, a chimeric polypeptide comprising the amino acidsequence set forth in SEQ ID NO: 11, or the amino acid sequence setforth in SEQ ID NO: 11 in the absence of one or both epitope tags istested in any one or more of the assays set forth in any of theexamples. In certain embodiments, a chimeric polypeptide in which theinternalizing moiety comprises an antibody or antigen-binding fragmentcomprising a light chain comprising the amino acid sequence of SEQ IDNO: 4 and comprising a heavy chain comprising the amino acid sequence ofSEQ ID NO: 2 is tested. In other embodiments, a chimeric polypeptide inwhich the internalizing moiety comprises an antibody or antigen-bindingfragment comprising a light chain comprising an amino acid sequence atleast 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 4 andcomprising a heavy chain comprising an amino acid sequence at least 95%,96%, 97%, 98%, or 99% identical to SEQ ID NO: 2 is tested.

Sequence Information

SEQ ID NO: 1 - amino acid sequence of the human MTM1 protein(NP_000243.1) MASASTSKYNSHSLENESIKRTSRDGVNRDLTEAVPRLPGETLITDKEVIYICPFNGPIKGRVYITNYRLYLRSLETDSSLILDVPLGVISRIEKMGGATSRGENSYGLDITCKDMRNLRFALKQEGHSRRDMFEILTRYAFPLAHSLPLFAFLNEEKFNVDGWTVYNPVEEYRRQGLPNHHWRITFINKCYELCDTYPALLVVPYRASDDDLRRVATFRSRNRIPVLSWIHPENKTVIVRCSQPLVGMSGKRNKDDEKYLDVIRETNKQISKLTIYDARPSVNAVANKATGGGYESDDAYHNAELFFLDIHNIHVMRESLKKVKDIVYPNVEESHWLSSLESTHWLEHIKLVLTGAIQVADKVSSGKSSVLVHCSDGWDRTAQLTSLAMLMLDSFYRSIEGFEILVQKKWISFGHKFASRIGHGDKNHTDADRSPIFLQFIDCVWQMSKQFPTAFEFNEQFLIIILDHLYSCRFGTFLFNCESARERQKVTERTVSLWSLINSNKEKFKNPFYTKEINRVLYPVASMRHLELWVNYYIRWNPRIKQQQPNPVEQRYMELLALRDEYIKRLEELQLANSAKLSDPPTSPSSPSQMMPHVQTHFSEQ ID NO: 2 - 3E10 Variable heavy chainEVQLVESGGGLVKPGGSRKLSCAASGFTFSNYGMHWVRQAPEKGLEWVAYISSGSSTIYYADTVKGRFTISRDNAKNTLFLQMTSLRSEDTAMYYCARRGLLLDYWGQGT TLTVSSSEQ ID NO: 3 - linker sequence “GS3” GGGGSGGGGSGGGGSSEQ ID NO: 4 - 3E10 Variable light chainDIVLTQSPASLAVSLGQRATISCRASKSVSTSSYSYMHWYQQKPGQPPKLLIKYASYLESGVPARFSGSGSGTDFHLNIHPVEEEDAATYYCQHSREFPWTFGGGTKLELKSEQ ID NO: 5 - human MTM1 nucleic acid sequence (NM_000252.2)agagggggcg gagcagggcc cggcagccga gcagcctggc aacggcggtg gcgcccggagcccgagagtt tccaggatgg cttctgcatc aacttctaaa tataattcac actccttggagaatgagtct attaagagga cgtctcgaga tggagtcaat cgagatctca ctgaggctgttcctcgactt ccaggagaaa cactaatcac tgacaaagaa gttatttaca tatgtcctttcaatggcccc attaagggaa gagtttacat cacaaattat cgtctttatt taagaagtttggaaacggat tcttctctaa tacttgatgt tcctctgggt gtgatctcga gaattgaaaaaatgggaggc gcgacaagta gaggagaaaa ttcctatggt ctagatatta cttgtaaagacatgagaaac ctgaggttcg ctttgaaaca ggaaggccac agcagaagag atatgtttgagatcctcacg agatacgcgt ttcccctggc tcacagtctg ccattatttg catttttaaatgaagaaaag tttaacgtgg atggatggac agtttacaat ccagtggaag aatacaggaggcagggcttg cccaatcacc attggagaat aacttttatt aataagtgct atgagctctgtgacacttac cctgctcttt tggtggttcc gtatcgtgcc tcagatgatg acctccggagagttgcaact tttaggtccc gaaatcgaat tccagtgctg tcatggattc atccagaaaataagacggtc attgtgcgtt gcagtcagcc tcttgtcggt atgagtggga aacgaaataaagatgatgag aaatatctcg atgttatcag ggagactaat aaacaaattt ctaaactcaccatttatgat gcaagaccca gcgtaaatgc agtggccaac aaggcaacag gaggaggatatgaaagtgat gatgcatatc ataacgccga acttttcttc ttagacattc ataatattcatgttatgcgg gaatctttaa aaaaagtgaa ggacattgtt tatcctaatg tagaagaatctcattggttg tccagtttgg agtctactca ttggttagaa catatcaagc tcgttttgacaggagccatt caagtagcag acaaagtttc ttcagggaag agttcagtgc ttgtgcattgcagtgacgga tgggacagga ctgctcagct gacatccttg gccatgctga tgttggatagcttctatagg agcattgaag ggttcgaaat actggtacaa aaagaatgga taagttttggacataaattt gcatctcgaa taggtcatgg tgataaaaac cacaccgatg ctgaccgttctcctattttt ctccagttta ttgattgtgt gtggcaaatg tcaaaacagt tccctacagcttttgaattc aatgaacaat ttttgattat aattttggat catctgtata gttgccgatttggtactttc ttattcaact gtgaatctgc tcgagaaaga cagaaggtta cagaaaggactgtttcttta tggtcactga taaacagtaa taaagaaaaa ttcaaaaacc ccttctatactaaagaaatc aatcgagttt tatatccagt tgccagtatg cgtcacttgg aactctgggtgaattactac attagatgga accccaggat caagcaacaa cagccgaatc cagtggagcagcgttacatg gagctcttag ccttacgcga cgaatacata aagcggcttg aggaactgcagctcgccaac tctgccaagc tttctgatcc cccaacttca ccttccagtc cttcgcaaatgatgccccat gtgcaaactc acttctgagg ggggaccctg gcaccgcatt agagctcgaaataaaggcga tagctgactt tcatttgggg catttgtaaa aagtagatta aaatatttgcctccatgtag aacttgaact aacataatct taaactcttg aatatgtgcc ttctagaatacatattacaa gaaaactaca gggtccacac ggcaatcaga agaaaggagc tgagatgaggttttggaaaa ccctgacacc tttaaaaagc agtttttgaa agacaaaatt tagatttaatttacgtcttg agaaatacta tatatacaat atatattttg tgggcttaat tgaaacaacattattttaaa atcaaagggg atatatgttt gtggaatgga ttttcctgaa gctgcttaacagttgctttg gattctctaa gatgaatcca aatgtgaaag atgcatgtta ctgccaaaaccaaattgagc tcagcttcct aggcattacc caaaagcaag gtgtttaagt aattgccagcttttatacca tcatgagtgg tgacttaagg agaaatagct gtatagatga gtttttcattatttggaaat ttaggggtag aaaatgtttt cccctaattt tccagagaag cctatttttatatttttaaa aaactgacag ggcccagtta aatatgattt gcatttttta aatttgccagttttattttc taaattcttt catgagcttg cctaaaattc ggaatggttt tcgggttgtggcaaacccca aagagagcac tgtccaagga tgtcgggagc atcctgctgc ttaggggaatgttttcgcaa atgttgctct agtcagtcca gctcatctgc caaaatgtag ggctaccgtcttggatgcat gagctattgc tagagcatca tccttagaaa tcagtgcccc agatgtacatgtgttgagcg tattcttgaa agtattgtgt ttatgcattt caatttcaat ggtgttggcttcccctcccc accccacgcg tgcataaaaa ctggttctac aaatttttac ttgaagtaccaggccgtttg ctttttcagg ttgttttgtt ttatagtatt aagtgaaatt ttaaatgcacagttctattt gctatctgaa ctaattcatt tattaagtat atttgtaaaa gctaaggctcgagttaaaac aatgaagtgt tttacaatga tttgtaaagg actatttata actaatatggttttgttttc aatgaattaa gaaagattaa atatatcttt gtaaattatt ttatgtcatagtttaattgg tctaccaagt aagacatctc aaatacagta gtataatgta tgaattttgtaagtataaga aattttatta gacattctct tactttttgt aaatgctgta aatatttcataaattaacaa agtgtcactc cataaaaaga aagctaatac taatagccta aaagattttgtgaaatttca tgaaaacttt ttaatggcaa taatgactaa agacctgctg taataaatgtattaactgaa acctaaaaaa aaaaaaaaaa aaSEQ ID NO: 6 - mouse MTM1 protein sequence (NP_064310.1)MASASASKYNSHSLENESIKKVSQDGVSQDVSETVPRLPGELLITEKEVIYICPFNGPIKGRVYITNYRLYLRSLETDSALILDVPLGVISRIEYMGGATSRGENSYGLDITCKDLRNLRFALKQEGHSRRDMFEILVKHAFPLAHNLPLFAFVNEEKFNVDGWTVYNPVEEYRRQGLPNHHWRISFINKCYELCETYPALLVVPYRTSDDDLRRIATFRSRNRLPVLSWIHPENKMVIMRCSQPLVGMSGKRNKDDEKYLDVIRETNKQTSKLMIYDARPSVNAVANKATGGGYESDDAYQNSELSFLDIHNIHVMRESLKKVKDIVYPNIEESHWLSSLESTHWLEHIKLVLTGAIQVADQVSSGKSSVLVHCSDGWDRTAQLTSLAMLMLDSFYRTIEGFEILVQKEWISFGHKFASRIGHGDKNHADADRSPIFLQFIDCVWQMSKQFPTAFEFNEGFLITVLDHLYSCRFGTFLFNCDSARERQKLTERTVSLWSLINSNKDKFKNPFYTKEINRVLYPVASMRHLELWVNYYIRWNPRVKQQQPNPVEQRYMELLALRDDYIKRLEELQLANSAKLADAPASTSSSSQMVPHVQTHFSEQ ID NO: 7 - mouse MTM1 nucleic acid sequence (NM_019926.2)ggtgagttcg ctttcttggc tgacctggct cggagccggg cattgcgggg atccaggattggaaaggttc caggatggct tctgcatcag catctaagta taattcacac tccttggagaatgaatccat taagaaagtg tctcaagatg gagtcagtca ggatgtgagt gagactgtccctcggctccc aggggagtta ctaattactg aaaaagaagt tatttacata tgtcctttcaatggccccat taagggaaga gtttacatca caaattatcg tctttattta agaagtttggaaacggattc tgctctaata cttgatgttc ctctgggtgt gatatcaaga attgaatatatgggaggcgc gactagtaga ggagaaaatt cctatggtct agatattact tgtaaagatttgagaaacct gaggtttgca ttgaagcaag aaggccacag cagaagagat atgtttgagatccttgtaaa acatgccttt cctctggcac acaatctgcc attatttgca tttgtaaatgaagagaagtt taacgtggat gggtggactg tttataatcc agttgaagaa tatagaaggcagggcctgcc caatcaccat tggaggataa gttttattaa caagtgctat gagctctgtgagacataccc tgctcttttg gtggttccct atcggacctc agatgatgat cttaggaggatcgcaacgtt tagatcccga aatcggcttc ctgtactgtc gtggattcac ccagaaaacaaaatggtcat tatgcgctgc agtcagcctc ttgtcggtat gagtggtaaa agaaataaagatgacgagaa atacctggat gtgatcaggg aaactaacaa acaaacttct aagctcatgatttatgatgc acgacccagt gtaaatgcag tcgccaacaa ggcaacagga ggaggatatgaaagtgatga cgcatatcaa aactcagaac tttccttctt agacattcat aatattcatgttatgcgaga atctttaaaa aaagtgaaag atattgttta tcccaacata gaagaatctcattggttgtc cagtttggag tctactcatt ggttagaaca tatcaagctt gttctgaccggtgccattca agtggcagac caagtgtctt caggaaagag ctcggtactt gtgcactgcagtgacggatg ggacaggacc gctcagctga catccttggc catgctgatg ttggacagcttctacagaac tattgaaggc tttgagatat tggtacagaa agagtggata agttttggccataaatttgc atctagaata ggtcatggtg ataaaaacca tgctgatgct gatcgatctcctatttttct tcagtttatt gactgtgtgt ggcagatgtc gaaacagttc cccacagcttttgagttcaa tgaaggcttt ttgattaccg ttttggatca tctgtatagc tgtcgatttggtactttctt attcaactgt gactcggctc gagaaagaca gaaacttaca gaaagaacagtttctctatg gtcgctaatt aacagcaata aagacaaatt caaaaacccc ttctatacaaaagaaatcaa tcgggttttg tatccagttg ccagcatgcg tcacttggaa ctgtgggtgaattattacat ccgatggaat cccagggtca agcagcaaca gcccaaccca gtggagcagcgttacatgga gcttttggcc ttgcgtgacg attatataaa gaggctcgag gaattgcagctggccaactc cgccaagctt gctgatgccc ccgcttcgac ttccagttcg tcacagatggtgccccatgt gcagacgcac ttctgagggg actcacttct ggcactgcac ttgaactctagataagtgaa atagctgact ctcattctgg gcatgtggac aaagtagatt taaagtgtctgcctccattt agaagttcaa ctaacatctt agacttttga gtatgtgcct tctgtaatacatatcacaag aaatcgatgg tgtccgtgtg gcaatcataa ggaaggagtc aagagggggttctggaaaat cctcatactt ttttttacaa agcacttttg caaagataaa acttaaatttaatttacctc tatataaatt ctacatatac agtatgtatt ttgtgggctt aattgaaatattattttaaa tccagggggg agatttgttt gcaaaatgta ttttcctcca gctgcttataacagttgctt tggattatct aaaattaatc caaatgtgaa agatgggtat tactgccaaagccaaattgc actctgcttc ttcagcaaat tccaagagca aggcgtttaa ataattgccaatttttattt taccataagt ggtaaggtaa aaagaaagat gaacatttca tcattttgaatttttgaaaa taaaaggttc tcccatcatt tttcaagaga agcacatttt tatattaagaaaaagtgata aggtttgatt tttttttccc tcaacattct cagctttgct ttctaaattatcccatgatt tttgtctaac actgagtcat actcaggttg aaggaaaccc ataaatagcactgtgcgagg agctggctgg cttctgctgc ttagaggaat atgttcgcaa acatgcctctagtcaattcg ccttatctgc tgaagtgtag gggcaccgcc ttgaatggat gagctatggctagagcatct ttctttacag taatgcccca ggtgtattct gtttatgtct ctctgtttaaatggtgtgcg tgcataaaaa cttgctctgc acattattac ttgaagtact gggcaatttgctttttcagg ttttttttca ttttgttttg tagtatgaaa tggaatttta aatgcacagttctatttgat atccgaacta attcatttag taaatatatt tgtaaaagct aaagttaaatcaattaatgt tttacagtga tttgtaaagg attatttata gctaatatgg ttttgttttcagtgaattaa gagagattac atttatcttt gtaaattatt ttatgtcata gcttaatggcctaccaaatg agacatctca aatataatag tataatgtat ggattttgta agtataaaaattattagata ttcgtttgct ttttgtaaac actgtaaata tttcataaat taaaatgtgtcactccataa gaagaaaaaa ctaatactaa tagttgacag gaattggtga aatttcatgaaaatattttc attgcaataa atattaaaag acctgctgSEQ ID NO: 8 - rat MTM1 protein sequence (NP_001013065.1)MASSSASDCDAHPVERESMRKVSQDGVRQDMSKSGPRLPGESAITDKEVIYICPFSGPVKGRLYITNYRLYLRSLETDLAPILDVPLGVISRIEKMGGVTSRGENSYGLDITCKDLRNLRFALKQEGHSRRDIFDVLTRHAFPLAYNLPLFAFVNEEKFKVDGWAIYNPVEEYRRQGLPDRHWRISFVNQRYELCDTYPALLVVPYRASDDDLRRVATFRSRNRIPVLSWIHPENRAAIMRCSQPLVGVGGKRSRDDERYLDIIRETNKQTSKLTIYDARPGVNAVANKATGGGYEGEDAYPHAELSFLDIHNIHVMRESLRRVRDIVYPHVEEAHWLSSLESTHWLEHIKLLLTGAIRVADKVASGLSSVLVHCSDGWDRTAQLTTLAMLMLDGFYRSIEGFEILVQKEWISFGHKFSSRIGHGDKNHADADRSPIFLQFIDCVWQMTKQFPTAFEFNECFLVAILDHLYSCRFGTFLLNCEAARERQRLAERTVSVWSLINSNKDEFTNPFYARESNRVIYPVTSVRHLELWVNYYIRWNPRIRQQQPHPMSEQ ID NO: 9 - rat MTM1 nucleic acid sequence (NM_001013047.1)gcgagcgcgt tggcaccagc ggcccccgga gtctcaggtt ccaggatggc gtcctcgtcagcctctgact gtgatgcaca ccccgtggag cgtgagtcca tgaggaaggt gtctcaagatggagtccgtc aggatatgag caagagtggg cctcgcctcc caggggaatc agccatcactgacaaggaag tcatctacat ttgtcccttc agcggccccg taaagggacg actttacatcaccaattacc gtctctacct gagaagtctg gagacggact tggctccgat tcttgacgtccccctaggcg tgatatcgag aatagagaaa atgggaggcg tgacgagtcg aggagagaattcctacggcc ttgatatcac ctgcaaagac ctgaggaacc tgaggttcgc tctgaagcaggaaggacaca gcaggaggga catctttgac gtcctcacca gacacgcctt ccccctggcttacaacctgc cgttgtttgc attcgtgaac gaggagaagt ttaaagtgga tggatgggcgatttacaacc cggttgaaga gtacagaagg cagggcctcc ccgatcgcca ttggcggataagtttcgtca atcagcgcta cgagctctgt gacacctacc ctgccctcct ggtcgtcccctaccgtgcgt ccgatgatga cctcagaaga gttgcaacct ttaggtccag aaaccggattcccgtgctgt cgtggatcca cccagagaac agggcggcga tcatgcggtg cagtcagcctctggttggtg tgggcgggaa gagaagcaga gatgatgaga gatacctgga catcatccgggaaaccaata agcagacctc gaagctcaca atttacgatg cgcggcccgg cgtcaatgcggtggccaaca aggcaacggg aggcggctat gagggcgagg acgcgtaccc tcacgcggagctctccttcc tggacatcca caacatccac gtgatgcggg aatccttacg gagggtgagggacatcgtgt acccccacgt ggaggaagct cattggctgt ccagcttgga gtccacccattggttagagc acatcaagct tctcctcact ggtgccatcc gggtcgcaga caaggtggcatcggggctga gttcagtcct cgtgcactgc agtgacggct gggaccggac ggctcaactgaccacgctgg ccatgctgat gctcgatggc ttctaccgca gcatcgaggg ctttgagatcctggtgcaga aggagtggat cagcttcgga cacaagtttt catctagaat tggccacggtgacaagaacc acgcggatgc cgaccgctcc ccgattttcc tgcagttcat cgactgcgtgtggcagatga cgaagcagtt ccccacagct ttcgagttca acgagtgctt cctggttgccatcttggatc acctgtacag ctgccggttc gggactttct tactaaactg tgaggcggcacgggagagac agagactcgc agaaaggacg gtgtctgtgt ggtccctgat caacagcaacaaagacgaat tcacaaaccc gttctacgca agggagagca accgcgtgat ctacccggtcaccagcgtgc gccacctgga actgtgggtg aattactaca tccggtggaa ccccaggatccggcagcagc agccccaccc catgtagcag cgatataatg agctcctggc cctgcgtgacgattacatca agaagctgga ggagctgcag ctggccacgc ccaccaagct cactgactcctccaccccgc cttccggttc cgcacagata gctccccgca tgcaaactca cttctgagggggttccgggc cccaaaccct gaataagtga cgtcaccaac ttccgttctg tgcgcttgtgcaaaggggat ataaagtctc cgcctctgtg tagaagtcga actaacaccc tagaaccttgtgtgacacgt gtgagtgtgc gccttttgtg acgtgtgagt gtgcgatttg tgtgacatgtgtgaatgtgt accctgtgtg atacgtgcaa gtgtgcgcct tgtgtaaagt tcgtgagtgtgcacctcctg taacatgttt tgcaaggaat ctactgcgct tgtgtgccag tcgtgagtacagagtagggg gggtcccgga aaaatcctca cactttttta caaagcgctt gtgcaaagattaaaattaaa ttatatcaat aattatataa attattataa ttatattgca aagattaaaaagttaaattt agtttacctc tatataaatc cagacataca taatatgtac tctgtgcgcttaattgaaac gttattttaa atccagaggg gagatttttt ttgtaaaatg gatttttcctccagccactt attttgcaaa gataaaaaag ttaaaataaa agttaaattt aattataaaaaaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaaaaaaaaaaaa aaaaaa SEQ ID NO: 10 - linker sequence “GSTS” GSTSGSGKSSEGKGSEQ ID NO: 11 - Fv3E10-GSTS-hMTM1DIVLTQSPASLAVSLGQRATISCRASKSVSTSSYSYMHWYQQKPGQPPKLLIKYASYLESGVPARFSGSGSGTDFHLNIHPVEEEDAATYYCQHSREFPWTFGGGTKLELKGGGGSGGGGSGGGSEVQLVESGGGLVKPGGSRKLSCAASGFTFSNYGMHWVRQAPEKGLEWVAYISSGSSTIYYADTVKGRFTISRDNAKNTLFLQMTSLRSEDTAMYYCARRGLLLDYWGQGTTLTVSSEQKLSEEL GSTSGSGKSSEGKGMASASTSKYNSHSLENESIKRTSRDGVNRDLTEAVPRLPGETLITDKEVIYICPFNGPIKGRVYITNYRLYLRSLETDSSLILDVPLGVISRIEKMGGATSRGENSYGLDITKDMRNLRFALKQEGHRRDMFEILTRYAFPLAHSLPLFAFLNEEKFNVDGWTVYNPVEEYRRQGLPNHHWRITFINKCYELCDTYPALLVVPYRASDDDLRRVATFRSRNRIPVLSWIHPENKTVIVCSQPLVGMSGKRNKDDKYLDVIRETNKQISKLTIYDARPSVNAVANKATGGGYESDDAYHNAELFFLDIHNIHVMRESLKKVKDIVYPNVEESHWLSSLESTHWLEHIKLVLTGAIQVADKVSSGKSVLVHCSDGWDRTAQLTLAMLMLDSFYRSIEGFEILVQKKWISFGHKFASRIGHGDKNHTDADRSPIFLQFIDCVWQMSKQFPTAFEFNEQFLIIILDHLYSCRFGTFLFNCESARERQKVTERTVLWSLINSNKEKFKNPFYTEINRVLYPVASMRHLELWVNYYIRWNPRIKQQQPNPVEQRYMELLALRDEYIKRLEELQLANSAKLSDPPTSPSSPSQMMPHVQTHFHHHHHH Note - in SEQ ID NO: 11 - linker sequences areunderlined and epitope tags are double underlinedIncorporation By Reference

All publications and patents mentioned herein are hereby incorporated byreference in their entirety as if each individual publication or patentwas specifically and individually indicated to be incorporated byreference.

While specific embodiments of the subject invention have been discussed,the above specification is illustrative and not restrictive. Manyvariations of the invention will become apparent to those skilled in theart upon review of this specification and the claims below. The fullscope of the invention should be determined by reference to the claims,along with their full scope of equivalents, and the specification, alongwith such variations.

I claim:
 1. A chimeric polypeptide comprising: (i) a myotubularin (MTM1) polypeptide, or a bioactive fragment thereof and (ii) an internalizingmoiety, wherein the chimeric polypeptide has phosphoinositidephosphatase activity; wherein said internalizing moiety is an antibodyor antigen-binding fragment thereof, wherein said antibody orantigen-binding fragment thereof is a monoclonal antibody 3E10, or avariant thereof that retains the cell penetrating activity of 3E10, oran antibody that binds the same epitope as 3E10, or an antigen-bindingfragment of any of the foregoing.
 2. The chimeric polypeptide of claim1, wherein the MTM1 polypeptide comprises an amino acid sequence havingat least 90% sequence identity to SEQ ID NO:
 1. 3. The chimericpolypeptide of claim 1, wherein the MTM1 polypeptide further comprisesone or more polypeptide portions that enhance one or more of in vivostability, in vivo half life, uptake/administration, and/orpurification.
 4. The chimeric polypeptide of claim 1, wherein theinternalizing moiety promotes transport of said chimeric polypeptideinto muscle cells.
 5. The chimeric polypeptide of claim 4, wherein saidantibody or antigen-binding fragment thereof is chimeric or humanized.6. The chimeric polypeptide of claim 4, wherein said antibody orantigen-binding fragment thereof comprises a light chain comprising anamino acid sequence having at least 98% sequence identity to SEQ ID NO:4 and a heavy chain comprising an amino acid sequence having at least98% sequence identity to SEQ ID NO:
 2. 7. The chimeric polypeptide ofclaim 1, wherein the antibody or antigen-binding fragment comprises aheavy chain variable domain (VH) comprising the amino acid sequence setforth in SEQ ID NO: 2 and a light chain variable domain (VL) comprisingthe amino acid sequence set forth in SEQ ID NO: 4, or which is ahumanized antibody or antigen-binding fragment thereof.
 8. The chimericpolypeptide of claim 1, wherein the internalizing moiety comprises ascFv.
 9. The chimeric polypeptide of claim 1, wherein the MTM1polypeptide or bioactive fragment thereof is conjugated or joined to theinternalizing moiety by a linker.
 10. The chimeric polypeptide of claim9, wherein the internalizing moiety is conjugated to the N-terminal orC-terminal amino acid of the MTM1 polypeptide.
 11. A compositioncomprising the chimeric polypeptide of claim 1, and a pharmaceuticallyacceptable carrier.
 12. The composition of claim 11, further comprisinga second agent which acts in an additive or synergistic manner fortreating myotubular myopathy.
 13. A nucleic acid construct, comprising anucleotide sequence that encodes the chimeric polypeptide of claim 1.14. A nucleic acid construct, comprising a nucleotide sequence thatencodes an MTM1 polypeptide or a bioactive fragment thereof, operablylinked to a nucleotide sequence that encodes an internalizing moiety,wherein the nucleic acid construct encodes a chimeric polypeptide havingphosphoinositide phosphatase activity; wherein said internalizing moietyis an antibody or antigen-binding fragment thereof, wherein saidantibody or antigen-binding fragment thereof is a monoclonal antibody3E10, or a variant thereof that retains the cell penetrating activity of3E10, or an antibody that binds the same epitope as 3E10, or anantigen-binding fragment of any of the foregoing.
 15. The nucleic acidconstruct of claim 14, wherein the antibody or antigen-binding fragmentcomprises a heavy chain variable domain (VH) comprising the amino acidsequence set forth in SEQ ID NO: 2 and a light chain variable domain(VL) comprising the amino acid sequence set forth in SEQ ID NO: 4, orwhich is a humanized antibody or antigen-binding fragment thereof. 16.The nucleic acid construct of claim 14, wherein the internalizing moietycomprises a scFv.
 17. A method of delivering a chimeric polypeptide intoa muscle cell, comprising contacting a muscle cell with a chimericpolypeptide, which chimeric polypeptide comprises an MTM1 polypeptide ora bioactive fragment thereof and an internalizing moiety which promotestransport into muscle cells, thereby delivering the chimeric polypeptideinto the muscle cell; wherein said internalizing moiety is an antibodyor antigen-binding fragment thereof, wherein said antibody orantigen-binding fragment thereof is a monoclonal antibody 3E10, or avariant thereof that retains the cell penetrating activity of 3E10, oran antibody that binds the same epitope as 3E10, or an antigen-bindingfragment of any of the foregoing.
 18. The method of claim 17, whereinthe MTM1 polypeptide comprises an amino acid sequence having at least90% sequence identity to SEQ ID NO: 1, or a bioactive fragment thereof.19. The method of claim 17, wherein the antibody or antigen-bindingfragment comprises a heavy chain variable domain (VH) comprising theamino acid sequence set forth in SEQ ID NO: 2 and a light chain variabledomain (VL) comprising the amino acid sequence set forth in SEQ ID NO:4, or which is a humanized antibody or antigen-binding fragment thereof.20. The method of claim 17, wherein the internalizing moiety comprises ascFv.